Methods, kits and compositions for the identification of nucleic acids electrostatically bound to matrices

ABSTRACT

This invention pertains to methods, kits and compositions suitable for the detection, identification and/or quantitation of nucleic acids which are electrostatically immobilized to matrices using non-nucleotide probes which sequence specifically hybridize to one or more target sequences of the nucleic acid but do not otherwise substantially interact with the matrix. Once the nucleic acid is immobilized, the detectable non-nucleotide probe/target sequence complex, formed before or after the immobilization of the nucleic acid, can be detected, identified or quantitated under a wide range of assay conditions as a means to detect, identify or quantitate the target sequence in the sample. Because it is reversibly bound, the non-nucleotide probe/target sequence can optionally be removed from the matrix for detecting, identifying or quantitating the target sequence in the sample. Because the non-nucleotide probe/target sequence is protected against degradation, it is another advantage of this invention that the sample can be treated with enzymes which degrade sample components, either before or after the nucleic acid is bound to the matrix, in order to “clean up” the sample (e.g. a complex biological sample such as a cell lysate) and thereby improve the detection, identification or quantitation of the target sequence in the sample. The methods, kits and compositions of this invention are therefore particularly well suited for the analysis, and particularly single point mutation analysis, in a particle assay, in an array assay, in a nuclease digestion/protection assay and/or in a line assay format. When utilized in combination with non-nucleotide “Beacon” probes, the invention is particularly well suited for use in a self-indicating assay format.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/456,73 filed on Dec. 8, 1999. This application claims thebenefit of U.S. Provisional Patent Application Serial No. 60/111,439filed on Dec. 8, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is related to the field of probe-based detection,analysis and quantitation of nucleic acids which are electrostaticallyimmobilized to matrices. The methods, kits and compositions of thisinvention are particularly well suited for the analysis, andparticularly single point mutation analysis, in a particle assay, in anarray assay, in a nuclease digestion/protection assay, in a line assayand/or in a self-indicating assay format.

[0004] 2. Description of the Related Art

[0005] Nucleic acid hybridization is a fundamental process in molecularbiology. Probe-based assays are useful in the detection, quantitationand analysis of nucleic acids. Nucleic acid probes have long been usedto analyze samples for the presence of nucleic acid from bacteria,eucarya, fungi, virus or other organisms and are also useful inexamining genetically-based disease states or clinical conditions ofinterest in single cells as well as in tissues.

[0006] Sample prep methods which describe the repetitive capture andrelease of target sequences to and from supports (e.g. magnetic beads)as a means to remove non-target polynucleotides, debris and impuritieswhich tend to introduce background in a hybridization assay are known inthe art (See: Collins et al., U.S. Pat. No. 5,750,338). Generally, thesample prep methods of Collins et al. can be used in most embodiments oftraditional hybridization assays provided however that the targetnucleic acid is first immobilized to a support and thereafter releasedfrom the support such that, when released, it is substantially free ofsample impurities, debris, and extraneous polynucleotides. The Collinset al. invention, however, requires that the probe or probes must beassociated with or capable of associating with the support under bindingconditions to thereby immobilize the nucleic acid of interest to thesupport (See: Collins et al. at col. 4, line 55 to col. 5, line 13).

[0007] A probe based sample prep method for removing contaminates priorto PCR reaction has been described by Goldin et al. (See: U.S. Pat. No.5,200,314). This process requires an analyte-capture probe having bothan analyte binding region and a first specific binding partner. Like theCollins et al. invention, the Goldin et al. invention requires that theanalyte-capture probe interact with the support as the specific meansthrough which the target sequence becomes immobilized.

[0008] Polycationic solid supports have been used for the analysis andpurification of nucleic acids, including the purification ofpolynucleotides from solutions containing contaminates (See: Arnold etal.; U.S. Pat. No. 5,599,667). Arnold et al. describe assays which usesolid supports as a means to separate polynucleotides, and hybridsthereof formed with a nucleotide probe, from unhybridized probe (See:Abstract to U.S. Pat. No. 5,599,667). The invention is premised upon “.. . the discovery that polycationic solid supports can be used toselectively adsorb nucleotide multimers according to their size(emphasis added), larger multimers being more tightly bound to thesupport than smaller ones.” (See: Col. 4, lines 39-44). The methods canalso be used to separate the nucleotide multimers from non-nucleotidicmaterial (See: Col. 5, lines 25-28).

[0009] A substantial limitation of the Arnold et al. invention is theinterplay which exists between the composition of the cationic solidsupport and the formulation of contacting solutions as well as theinterplay between two or more of the contacting solutions (See: Col. 7,line 24 to Col. 8, line 32) which are required to discriminate betweennucleotide multimers (See: Col. 8, lines 39-41). An example of alaborious protocol for arriving at a proper cation density for a solidsupport can be found at col. 9, lines 36-52 and the method fordetermining the buffer concentration suitable for separatingpolynucleotides and nucleotide probes can be found at col. 9, lines53-63. Similarly, the separation solution must be carefully designed(See: Col. 10. lines 9-12), presumably using the laborious method oftrial and error as described for determining the cation density of thesolid support. This requirement for substantial optimization of assayconditions within a very narrow operating range results becauseelectrostatic immobilization of nucleic acid is a relativelynon-specific process and therefore it is difficult to electrostaticallyimmobilize a negatively charged target nucleic acid to a cationicsurface without the positively charged matrix also exhibiting a strongaffinity for the negatively charged nucleic acid probe. Since theseparation of nucleotide multimers (nucleotide probe/target hybrids fromexcess nucleotide probe) occurs within a narrow range of conditions,which may not necessarily be optimal for the discrimination ofhybridization, the hybrids still immobilized according to the Arnold etal. invention may not be truly indicative of the presence of a targetsequence. Consequently, the applicability of the assays of Arnold et al.are of limited practical utility.

[0010] An invention related to achieving nucleic acid has recently beendescribed (See: Gerdes et al.; WO98/46797). Gerdes et al. use highlyelectropositive solid phase materials to capture nucleic acids (See. p.5, line 24 to p. 6, line 14) for repetitive analyses. However, asubstantial limitation of the Gerdes et al. invention is that thenucleic acid must be irreversibly bound to the highly electropositivesolid phase material.

[0011] Methods for the high throughput screening for sequences orgenetic alterations in nucleic acid have been described (See: Shuber, A.P.; U.S. Pat. No. 5,834,181). Shuber describes the analysis of arrays ofimmobilized nucleic acids, and suggests immobilization of the nucleicacid to nitocellulose or a charged nylon membrane (See: col. 6, lines41-64). Suggested purine and pyrimidine containing polymers which may beused for analyzing immobilized nucleic acid include peptide nucleic acid(See: col. 5, lines 15-20), but the polymers must necessarily be taggedor labeled since the detection methods rely on a tag or label beingincorporated into the polymer (See: col. 8, line 58 to col. 9, line 3).The assays of Shuber require a perfect complement between probe andtarget sequence (See: col. 8, lines 52-57). In order to achieve properdiscrimination, a laborious empirical process of trial and error isdescribed for assay optimization (See: col. 7, line 16 to col. 8).Conditions which require optimization of specific and non-specifichybridization include the concentration of polymer, the temperature ofhybridization, the salt concentration, and the presence or absence ofunrelated nucleic acid (See: col. 8, lines 15-18).

[0012] Shuber does not expressly suggest performing a probe-basedhybridization assay on an electrostatically immobilized nucleic acid andspecifically does not describe or teach the analysis ofelectrostatically immobilized nucleic acid using a non-nucleotide probesuch as a peptide nucleic acid. Furthermore, Shuber does not suggest,disclose or teach any advantages, such the ability to work within abroad range of assay conditions, of performing a peptide nucleicacid-based analysis of nucleic acid electrostatically immobilized to amatrix.

[0013] Pluskal et al. describe a comparison of DNA and peptide nucleicacid (PNA) probe-based analysis of nucleic acid which has beenirreversibly crosslinked to charged nylon membrane (See: Pluskal et al.,American Society for Biochemistry, 85th Annual Meeting, Washington, DC,May 1994). Pluskal et al. teach that while PNA probes can be used todetect the irreversibly immobilized nucleic acid under standardhybridization conditions, PNA works very well under highly stringenthybridization and washing conditions (See: The Section Entitled“Discussion”). Pluskal et al. also teach the use of 1% BSA as a blockingagent to reduce non-specific binding of the probe to the membrane (See:Section Entitled “Discussion”). Because the nucleic acid of Pluskal etal. has been irreversibly crosslinked to the nylon membrane, highlystringent hybridization and washing conditions can be applied to themembrane without reducing the amount of target nucleic acid present onthe support and available for analysis. Pluskal et al. thereforedemonstrate a rational for irreversibly linking the nucleic acid to beanalyzed to the support and using a blocking agent when performing a PNAprobe-based analysis using a charged nylon membrane.

[0014] Methods for the protection of nucleic acid sequences fromnuclease degradation/digestion by hybridizing a nucleic acid analogthereto have been described (See: Stanley et al.; U.S. Pat. No.5,861,250). The methods and compositions described in Stanley et al. areparticularly well suited for ““cleaning up” a nucleic acid sample bydegrading all nucleic acid present except the target sequence, . . . ”(See: Stanley et al. at col. 7, lines 14-18). Stanley et al. describeseveral means for separating hybridized nucleic acid analog fromnon-hybridized nucleic acid analog, including ion exchangechromatography (See: Col. 6, lines 62-64), but they do not describe thesimple electrostatic immobilization of the target sequence or nucleicacid analog/target sequence complex to a matrix as means to separate thehybridized nucleic acid analog from non-hybridized nucleic acid analogor otherwise separate the nucleic acid analog/target sequence complexfrom the other components of a sample.

[0015] Methods and apparatus for the electroactive transport andfixation of nucleic acids for analysis have been described (See: Helleret al., U.S. Pat. No. 5,849,486). However, this invention requireshighly sophisticated instrumentation and devices to transport, fixand/or analyze a sample.

[0016] Though van den Engh does not discuss the detection of complexmacromolecules such as nucleic acids, fluorescent reporter beads andmethods for detecting the presence or determining the concentration offluid bulk analytes such as pH, oxygen saturation and ion content areknown in the art (See: van den Engh et al., U.S. Pat. No. 5,747,349).According to van den Engh, “Reporter beads are added to a fluid sampleand the analyte concentration is determined by measuring fluorescence ofindividual beads, for example in a flow cytometer” (See: Abstract ofU.S. Pat. No. 5,747,349). The beads of van den Engh et al. comprise asubstrate bead having a plurality of fluorescent reporter moleculesimmobilized thereon wherein the fluorescent reporter molecules comprisea fluorescent molecule whose fluorescent properties are a function ofthe concentration of the particular analyte whose presence orconcentration is to be determined (See: U.S. Pat. No. 5,747,349 at col.3, lns. 29-46). Thus, the beads of van den Engh are inherentlyfluorescent and not the analytes or derivatives thereof.

[0017] Recently, compositions containing at least one bead conjugated toa solid support and further conjugated to at least one macromoleculehave been described in the art (See: Lough et al., PCT/US97/20194).Claimed advantages of Lough et al. include increased surface area forthe immobilization of biological particles or macromolecules as comparedto flat surfaces as well as the ability to use one chemistry for theimmobilization of the macromolecule to the bead and a differentchemistry to attach the bead to the support. Lough et al. definemacromolecules to include nucleic acids (See: p. 7, lns. 10-17) andfurther define peptide nucleic acids (PNA) as being analogs of nucleicacids (See: p. 8, lns. 4-9). The invention of Lough et al. is primarilydirected to analysis of immobilized macromolecules. Curiously however, aprobe-based assay is not described as a detection method but ratherLough et al. focus on direct analysis of the immobilized macromoleculebe means such as MALDI-TOF mass spectrometry. Apart from apparentlybeing considered by Lough et al. to be an analog of a nucleic acid, PNAis not otherwise mentioned in the disclosure and no examples areprovided which demonstrate that PNA is suitable for the practice of theinvention.

[0018] Despite its name, Peptide Nucleic Acid (PNA) is neither apeptide, a nucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) isa non-naturally occurring polyamide which can hybridize to nucleic acid(DNA and RNA) with sequence specificity (See: U.S. Pat. Nos. 5,539,082,5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,461 aswell as Eghohm et al., Nature 365: 566-568 (1993)). Being anon-naturally occurring molecule, unmodified PNA is not known to be asubstrate for the enzymes which are known to degrade peptides or nucleicacids. Therefore, PNA should be stable in biological samples, as well ashave a long shelf-life. Unlike nucleic acid hybridization which is verydependent on ionic strength, the hybridization of a PNA with a nucleicacid is fairly independent of ionic strength and is favored at low ionicstrength, conditions which strongly disfavor the hybridization ofnucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). Theeffect of ionic strength on the stability and conformation of PNAcomplexes has been extensively investigated (Tomac et al., J. Am. Chem.Soc. 118:55 44-5552 (1996)). Sequence discrimination is more efficientfor PNA recognizing DNA than for DNA recognizing DNA (Egholm et al.,Nature, at p. 566). However, the advantages in point mutationdiscrimination with PNA probes, as compared with DNA probes, in ahybridization assay, appears to be somewhat sequence dependent (Nielsenet al., Anti-Cancer Drug Design 8:53-65, (1993) and Weiler et al., Nucl.Acids Res. 25: 2792-2799 (1997)).

[0019] Because the nucleic acids of a complex sample, such as a celllysate or PCR reaction mixture, can be concentrated and also partiallypurified by immobilization to supports, probe-based hybridization assayscould be simplified if the presence of a target nucleic acid could bespecifically detected while the target nucleic acid remained supportbound; particularly if the conditions for the treatment, analysis and/ordetection of the target sequence were operable within a broad range sothat assay conditions did not require substantial and laboriousoptimization. The ability to perform such analyses using a flowcytometer, an array, a nuclease digestion/protection assay, a lineassay, a self-indicating assay or in some combination of these assayformats would be particularly beneficial.

SUMMARY OF THE INVENTION

[0020] This invention pertains to methods, kits and compositionssuitable for the detection, identification and/or quantitation ofnucleic acids which are electrostatically immobilized to matrices usingnon-nucleotide probes which sequence specifically hybridize to one ormore target sequences of the nucleic acid but do not otherwisesubstantially interact with the matrix. Once the nucleic acid isimmobilized, the detectable non-nucleotide probe/target sequencecomplex, formed before or after the immobilization of the nucleic acid,can be detected, identified or quantitated under a wide range of assayconditions as a means to detect, identify or quantitate the targetsequence in the sample. Because it is reversibly bound, thenon-nucleotide probe/target sequence can optionally be removed from thematrix for detecting, identifying or quantitating the target sequence inthe sample. Because the non-nucleotide probe/target sequence isprotected against degradation, it is another advantage of this inventionthat the sample can be treated with enzymes which degrade samplecomponents, either before or after the nucleic acid is bound to thematrix, in order to “clean up” the sample (e.g. a complex biologicalsample such as a cell lysate) and thereby improve the detection,identification or quantitation of the target sequence in the sample.Consequently, the methods, kits and compositions of this invention havesubstantial advantages over all previously known or described methods,kits or compositions because they facilitate the simple processingand/or analysis of samples, and particularly complex biological samples,under a wide range of assay conditions.

[0021] In one embodiment, this invention is related to a compositioncomprising a nucleic acid, having at least one target sequence, which iselectrostatically bound to a matrix under suitable electrostatic bindingconditions. The composition further comprises a detectable, but notnecessarily labeled, non-nucleotide probe having a probing nucleobasesequence which is sequence specifically hybridized to at least a portionof the target sequence.

[0022] In another embodiment, this invention pertains to methods for thedetection, identification or quantitation of a target sequence in asample containing nucleic acid. One exemplary method comprisescontacting a sample with a matrix and at least one non-nucleotide probewherein the nucleic acid in the sample will electrostatically bind tothe matrix under suitable electrostatic binding conditions.Additionally, the non-nucleotide probe will hybridize, under suitablehybridization conditions, to at least a portion of the target sequence,if present in the sample. The method further comprises detecting,identifying or quantitating the non-nucleotide probe/target sequencehybrid as a means to detect, identify or quantitate the target sequencein the sample.

[0023] In still another embodiment, this invention pertains to multiplexmethods for the detection, identification or quantitation of two or moretarget sequences of one or more nucleic acid molecules which may bepresent in the same sample. One exemplary method comprises contacting asample with a matrix and two or more independently detectablenon-nucleotide probes wherein the nucleic acid present in the samplewill electrostatically bind to the matrix under suitable electrostaticbinding conditions. Additionally, the two or more independentlydetectable non-nucleotide probes will hybridize, under suitablehybridization conditions, to at least a portion of the target sequenceswith which each probe is designed to hybridize if present in the nucleicacid of the sample. Consequently, if a particular target sequence iselectrostatically immobilized to the matrix, the independentlydetectable non-nucleotide probe designed to hybridized to thatparticular target sequence will become concentrated on the matrix and beavailable for detection. Therefore, the method further comprisesdetecting, identifying or quantitating each unique independentlydetectable non-nucleotide probe/target sequence hybrid which iselectrostatically bound to said matrix as a means to detect, identify orquantitate each unique target sequence sought to be detected in thesample and in the same assay. Optionally, the unique independentlydetectable non-nucleotide probe/target sequence hybrid is released fromthe matrix by adjustment of conditions outside the range required forelectrostatic binding and thereby facilitates detection of the unboundnon-nucleotide probe/target sequence hybrid, or just the detectableprobe, as the means to detect, identify or quantitate the targetsequence in the sample.

[0024] In still a further embodiment, this invention takes advantage ofthe stability of nucleic acid analog/nucleic acid complexes (See:Stanley et al.; U.S. Pat. No. 5,861,250) to thereby further improveassay performance and/or otherwise decrease the labor or complexity ofsample preparation. One exemplary method comprises contacting the samplewith at least one non-nucleotide probe wherein the non-nucleotide probewill hybridize, under suitable hybridization conditions, to at least aportion of the target sequence if present in the sample. The sample isalso contacted with a matrix wherein the nucleic acid molecule willelectrostatically bind to a matrix under suitable electrostatic bindingconditions. Either before or after immobilization to the matrix, thesample containing the non-nucleotide probe/target sequence complex iscontacted with one or more enzymes capable degrading samplecontaminates, possibly including the nucleic acid molecule but not thenon-nucleotide probe/target sequence complex. The method furthercomprises detecting, identifying or quantitating the non-nucleotideprobe/target sequence hybrid as a means to detect, identify orquantitate the target sequence in the sample. Optionally, the detectablenon-nucleotide probe/target sequence hybrid is released from the matrixby adjustment of conditions outside the range required for electrostaticbinding and thereby facilitates detection of the unbound non-nucleotideprobe/target sequence hybrid, or just the detectable probe, as the meansto detect, identify or quantitate the target sequence in the sample.

[0025] In yet another embodiment, this invention relates to a method forthe detection, identification or quantitation of a target sequence of anucleic acid molecule electrostatically immobilized at a location on anarray wherein the array comprises nucleic acid moleculeselectrostatically bound at unique locations. One exemplary methodcomprises contacting the array with at least one non-nucleotide probe,wherein the non-nucleotide probe will hybridize, under suitablehybridization conditions, to at least a portion of the target sequenceif present on the array. The non-nucleotide probe/target sequencecomplex electrostatically bound at a location on said array is thendetected, identified or quantitated as the means to determine thepresence, absence or amount of target sequence present at said arraylocation. It is an advantage of the invention that one or more enzymescapable degrading sample contaminates, including the nucleic acid targetmolecule but not the non-nucleotide probe/target sequence complex, canalso be added before analysis of the array to thereby improve theperformance of the array assay by degrading sample contaminates whichmight otherwise lead to false positive results. Optionally, thedetectable non-nucleotide probe/target sequence hybrids can be releasedfrom the matrix by adjustment of conditions outside the range requiredfor electrostatic binding and thereby facilitates detection of theunbound non-nucleotide probe/target sequence hybrid, or just thedetectable probe, as the means to detect, identify or quantitate targetsequence in the sample. If the non-nucleotide probes are independentlydetectable, the analysis of the matrix can proceed in a multiplexformat.

[0026] In yet a further embodiment, this invention is directed to amethod for the detection, identification or quantitation of a targetsequence of a nucleic acid molecule which may be present in any of twoor more samples of interest. The method comprises mixing each of the twoor more samples of interest with at least one non-nucleotide probe,under suitable hybridization conditions. Next a matrix is contacted,under suitable electrostatic binding conditions, with at least a portionof each of the two or more samples to thereby electrostaticallyimmobilize the nucleic acid components of each sample to the matrix,each at a unique location, and thereby create a matrix array of samples.The non-nucleotide probe/target sequence complex electrostatically boundat a location on said array is then detected, identified or quantitatedas the means to determine the presence, absence or amount of targetsequence present at said array location. It is an advantage of theinvention that one or more enzymes capable degrading samplecontaminates, possibly including the nucleic acid target molecule butnot the non-nucleotide probe/target sequence complex, can also be addedbefore analysis of the array to thereby improve the performance of thearray assay by degrading sample contaminates which might otherwise leadto false positive results. Optionally, the detectable non-nucleotideprobe/target sequence hybrids can be released from the matrix byadjustment of conditions outside the range required for electrostaticbinding and thereby facilitates detection of the unbound non-nucleotideprobe/target sequence hybrid, or just the detectable probe, as the meansto detect, identify or quantitate target sequence in the sample. If thenon-nucleotide probes are independently detectable, the analysis of thematrix can proceed in a multiplex format.

[0027] In yet another embodiment, this invention is directed to kitssuitable for performing an assay which detects the presence, absence ornumber of target sequences present in a sample. The kits of thisinvention comprise a matrix and one or more non-nucleotide probes andoptionally one or more other reagents or compositions which are selectedto perform an assay of this invention or otherwise simplify theperformance of an assay used to detect, identify or quantitate a targetsequence in a sample.

[0028] The compositions, methods and kits of this invention areparticularly useful for the detection, identification and/or enumerationof bacteria and eucarya (e.g. pathogens) in food, beverages, water,pharmaceutical products, personal care products, dairy products orenvironmental samples. The analysis of preferred non-limiting beveragesinclude soda, bottled water, fruit juice, beer, wine or liquor products.Suitable compositions, methods and kits will be particularly useful forthe analysis of raw materials, equipment, products or processes used tomanufacture or store food, beverages, water, pharmaceutical products,personal care products dairy products or environmental samples.

[0029] Additionally, the compositions, methods and kits of thisinvention are particularly useful for the detection of bacteria andeucarya (e.g. pathogens) in clinical samples and clinical environments.Suitable compositions, methods and kits will be particularly useful forthe analysis of clinical specimens, equipment, fixtures or products usedto treat humans or animals.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1A and 1B are computer generated negatives of the image of aphotograph of tubes from an experiment using PCR to amplify a nucleicacid comprising a target sequence to which a Linear Beacon hybridizes togenerate detectable signal.

[0031]FIGS. 2A and 2B are computer generated negatives or the image of aphotograph of the same polyacrylamide gel used to analyze the content ofthe tubes shown in FIGS. 1A and 1B, before (FIG. 2A) and after (FIG. 2B)ethidium bromide staining.

[0032]FIG. 3 is a computer generated negative of an image of aphotograph of tubes from an experiment using PCR to generate differentamplicons having a point mutation of a target sequence to which a LinearBeacon hybridizes to generate detectable signal.

[0033]FIGS. 4A and 4B are computer generated negatives of an image of aphotograph of the same polyacrylamide gel used to analyze the content oftubes shown in FIG. 3, before (FIG. 4A) and after (FIG. 4B) ethidiumbromide staining.

[0034]FIG. 5 is a computer generated negative of an image of aphotograph of tubes from an experiment used to determine the range ofionic strength suitable for electrostatic immobilization of probes,target nucleic acids and probe/target nucleic acid complexes.

[0035]FIGS. 6A and 6B are images of the same GAPS coated microscopeslide containing spotted samples, before (6A) and after (6B) washing toremove material not otherwise electrostatically immobilized.

DETAILED DESCRIPTION OF THE INVENTION

[0036] 1. Definitions:

[0037] a. As used herein, the term “nucleobase” shall include thosenaturally occurring and those non-naturally occurring heterocyclicmoieties commonly known to those who utilize nucleic acid technology orutilize peptide nucleic acid technology to thereby generate polymerswhich can sequence specifically hybridize to nucleic acids.

[0038] b. As used herein, the term “nucleobase sequence” is any segmentof a polymer which comprises nucleobase containing subunits.Non-limiting examples of suitable polymers or polymers segments includeoligonucleotides, oligoribonucleotides, peptide nucleic acids andanalogs or chimeras thereof.

[0039] c. As used herein, the term “target sequence” is the nucleobasesequence of a nucleic acid molecule of interest which is sought to bedetected in an assay and to which at least a portion of the probingnucleobase sequence of the non-nucleotide probe is intended tohybridize. The target sequence may comprise a subset of the nucleic acidmolecule or may be the entire nucleic acid molecule of interest.

[0040] d. As used herein, the terms “label” and “detectable moiety”shall be interchangeable and shall refer to moieties which can beattached to a non-nucleotide probe, antibody or antibody fragment tothereby render the non-nucleotide probe, antibody or antibody fragmentdetectable by an instrument or method.

[0041] e. As used herein, the term “non-nucleotide probe” shall mean apolymer which is not a polynucleotide but which comprises a probingnucleobase sequence which is designed to hybridize to at least a portionof the target sequence. A preferred non-limiting example of anon-nucleotide probe is a peptide nucleic acid (PNA) probe.

[0042] f. As used herein, the term “peptide nucleic acid” or “PNA” shallbe defined as a non-nucleotide polymer comprising two or more PNAsubunits (residues), including any of the compounds referred to orclaimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675,5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,461 (all of whichare herein incorporated by reference). The term “peptide nucleic acid”or “PNA” shall also apply to polymers comprising two or more subunits ofthose nucleic acid mimics described in the following publications:Diderichsen et al., Tett. Lett. 37: 475-478 (1996); Fujii et al.,Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordan et al., Bioorg. Med.Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett. Lett. 36: 6941-6944(1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994);Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1: 539-546; Lowe etal., J. Chem. Soc. Perkin Trans. 11: 547-554 (1997); Lowe et al., J.Chem. Soc. Perkin Trans. 11:5 55-560 (1997); Petersen et al., Bioorg.Med. Chem. Lett. 6: 793-796 (1996); Diederichsen, U., Bioorganic & Med.Chem. Lett., 8: 165-168 (1998); Cantin et al., Tett. Lett., 38:4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997);Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997) and WIPO patentapplication WO96/04000 by Shah et al. and entitled “Peptide-basednucleic acid mimics (PENAMs)”.

[0043] In preferred embodiments, a PNA is a polymer comprising two ormore subunits of the formula:

[0044] wherein, each J is the same or different and is selected from thegroup consisting of H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I. EachK is the same or different and is selected from the group consisting ofO, S, NH and NR¹. Each R¹ is the same or different and is an alkyl grouphaving one to five carbon atoms which may optionally contain aheteroatom or a substituted or unsubstituted aryl group. Each A isselected from the group consisting of a single bond, a group of theformula; —(CJ₂)_(s)— and a group of the formula; —(CJ₂)_(s)C(O)—,wherein, J is defined above and each s is an whole number from one tofive. The whole number t is 1 or 2 and the whole number u is 1 or 2.Each L is the same or different and is independently selected from thegroup consisting of J. adenine, cytosine, guanine, thymine, uridine,5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil,2-thiothymidine, other naturally occurring nucleobase analogs, othernon-naturally occurring nucleobases, substituted and unsubstitutedaromatic moieties, biotin, fluorescein and dabcyl. In the most preferredembodiment, a PNA subunit consists of a naturally occurring ornon-naturally occurring nucleobase attached to the aza nitrogen of theN-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

[0045] 2. Description

[0046] I. General:

[0047] Probes:

[0048] The probes used for the practice of this invention arenon-nucleotide probes which at a minimum comprise a probing nucleobasesequence designed to hybridize to at least a portion of a targetsequence sought to be detected in a probe-based hybridization assay. Thenon-nucleotide probes comprise a sufficiently neutral or positivelycharged backbone such that they exhibit little or no affinity for thematrix under a broad range of assay conditions including substantialvariations in pH, buffer ionic strength, detergent concentration and/orchemical denaturant concentration. This broad range of conditions underwhich little or no interaction occurs between the non-nucleotide probeand the matrix is a substantial advantage over the previous methods suchas those of Arnold et al. (U.S. Pat. No. 5,599,667) which requiredsubstantial condition optimization to achieve discrimination of betweenthe non-specific binding of the nucleotide probe and the target nucleicacid (or nucleotide probe/target nucleic acid complex) to the matrix.

[0049] It is still another advantage of this invention that substantialchanges in the ionic strength of the assay have little effect on the Tmof the non-nucleotide probe/target sequence hybrid (See: (Egholm et alNature 365: 566-568 (1993) and Tomac et al., J. Am. Chem. Soc. 118:5544-5552 (1996)). Consequently, this lack of sensitivity of the hybrid tochanges in ionic strength also broadens the range of possible assayconditions.

[0050] The non-nucleotide probes may be labeled with a detectable moietyor may be unlabeled provided however that the non-nucleotideprobe/target sequence hybrid is detectable when the probe is unlabeled.The preferred non-nucleotide probes are PNA probes. Preferred labelednon-nucleotide probes are non-nucleotide “Beacon” probes (See: theSection entitled “Non-Nucleotide “Beacon” Probes, below) because theyare self-indicating. By self-indicating we mean that the probes changedetectable properties upon hybridization to a target sequence andthereby reduce or eliminate the requirement for the removal of excessprobe. In one embodiment, the self-indicating probes of this inventionwill rely on a change in fluorescence which can be observed with the eyeor otherwise detected and/or quantitated with a fluorescence instrument.

[0051] Because it is an important feature of this invention that thenon-nucleotide probes do not substantially interact with the matrix, thenon-nucleotide probes of this invention may also be designed, byappropriate modification, to have a particular net charge. For example,certain choices of labels might cause the non-nucleotide probe to have anet negative charge (See: Example 9). However, the net charge of theprobe can be changed by adding one or more positively charged moieties,such as by linking one or more of compounds 7 or 8 as described byGildea et al., Tett. Lett. 39: 7255-7258 (1998). By the alteration ofnet charge, the probes can be designed to have any combination ofdesired labels and still not exhibit an affinity for the matrix.

[0052] Unlabeled Non-Nucleotide Probes:

[0053] The non-nucleotide probes used for the practice of this inventionneed not be labeled with a detectable moiety to be operable within themethods of this invention. When using the non-nucleotide probes it ispossible to detect the non-nucleotide probe/nucleic acid complex formedby hybridization of the probing nucleobase sequence of the probe to thetarget sequence using an antibody to the non-nucleotide probe/nucleicacid hybrid (complex). As a non-limiting example, a PNA/nucleic acidcomplex could be detected using an antibody which specifically interactswith the complex under suitable antibody binding conditions. Suitableantibodies to PNA/nucleic acid complexes and methods for theirpreparation and use are described in WIPO Patent Application WO95/17430as well as U.S. Pat. No. 5,612,458, herein incorporated by reference.

[0054] The antibody/PNA/nucleic acid complex formed by interaction ofthe α-PNA/nucleic acid antibody with the PNA/nucleic acid complex can bedetected by several methods. For example, the α-PNA/nucleic acidantibody could be labeled with a detectable moiety. Suitable detectablemoieties (labels) are described herein. Thus, the presence, absence orquantity of the detectable moiety is correlated with the presence,absence or quantity of the antibody/PNA/nucleic acid complex and thetarget sequence sought to be identified. Alternatively, theantibody/PNA/nucleic acid complex is detected using a secondary antibodywhich is labeled with a detectable moiety. Typically the secondaryantibody specifically binds to the α-PNA/nucleic acid antibody undersuitable antibody binding conditions. Thus, the presence, absence orquantity of the detectable moiety is correlated with the presence,absence or quantity of the antibody/antibody/PNA/nucleic acid complexand the target sequence sought to be identified. As used herein, theterm antibody shall include antibody fragments which specifically bindto other antibodies or other antibody fragments.

[0055] Probing Nucleobase Sequence:

[0056] The probing nucleobase sequence of a non-nucleotide probe usedfor the practice of this invention is the sequence recognition portionof the construct. Therefore, the probing nucleobase sequence is designedto hybridize to at least a portion of the target sequence since it maybe preferable to use two or more probes designed to hybridize to theentire target sequence (See for example: European Patent Applicationentitled “Method of identifying a nucleic acid using triple helixformation of adjacently annealed probes”; EP-A-849-363 as well as WIPOpatent application No. WO99/55916 entitled “Methods, Kits andCompositions for Detecting and Quantitating Target Sequences”.Preferably, the probing nucleobase sequence hybridizes to the entiretarget sequence. Detection of non-nucleotide probe hybridization to thetarget sequence can be correlated with the presence, absence or amountof target sequence present in a sample.

[0057] The probing nucleobase sequence of a non-nucleotide probe willpreferably be exactly complementary to all or a portion of the targetsequence. Alternatively, a substantially complementary probingnucleobase sequence might be used since it has been demonstrated thatgreater sequence discrimination can be obtained when utilizing probeswherein there exists one or more point mutations (base mismatch) betweenthe probe and the target sequence (See: Guo et al., Nature Biotechnology15:331-335 (1997)).

[0058] With due consideration to the requirements of a non-nucleotideprobe for the assay format chosen and the target sequence sought to bedetected, the probing nucleobase sequence will generally be chosen suchthat a stable complex is formed with all or a portion of the targetsequence, under suitable hybridization conditions. Generally however,the non-nucleotide probes suitable for the practice of this invention,will generally have a probing nucleobase sequence in the range of 5-50subunits. More preferably, the probing nucleobase sequence will be inthe range of 7-25 subunits in length and most preferably in the range of12-20 subunits in length.

[0059] PNA Synthesis:

[0060] Methods for the chemical assembly of PNAs are well known (See:U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336,5,773,571 or 5,786,571, herein incorporated by reference). Chemicals andinstrumentation for the support bound automated chemical assembly ofpeptide nucleic acids are now commercially available. Chemical assemblyof a PNA is analogous to solid phase peptide synthesis, wherein at eachcycle of assembly the oligomer possesses a reactive alkyl amino terminuswhich is condensed with the next synthon to be added to the growingpolymer. Because standard peptide chemistry is utilized, natural andnon-natural amino acids are routinely incorporated into a PNA oligomer.Because a PNA is a polyamide, it has a C-terminus (carboxyl terminus)and an N-terminus (amino terminus). For the purposes of the design of ahybridization probe suitable for antiparallel binding to the targetsequence (the preferred orientation), the N-terminus of the probingnucleobase sequence of the PNA probe is the equivalent of the5′-hydroxyl terminus of an equivalent DNA or RNA oligonucleotide.

[0061] PNA Labeling:

[0062] Labeling of a PNA is analogous to peptide labeling. Because thesynthetic chemistry of assembly is essentially the same, any methodcommonly used to label a peptide can usually be adapted for use inlabeling a PNA. Thus, PNAs may be labeled with numerous detectablemoieties. Generally, any detectable moiety which can be linked to anucleic acid or peptide can be linked to a PNA.

[0063] Typically, the N-terminus of the PNA is labeled by reaction witha moiety having a carboxylic acid group or activated carboxylic acidgroup. One or more spacer moieties can be introduced between the labeledmoiety and the PNA oligomer. Generally, the spacer moiety isincorporated prior to performing the labeling reaction. However, thespacer may be embedded within the label and thereby be incorporatedduring the labeling reaction. Specialized reagents can be attached tothe PNA. For example, a terminal arylamine moiety can be generated bycondensing a suitably protected 4-aminobenzoic acid derivative with theamino terminus of the PNA oligomer.

[0064] In one embodiment, the C-terminal end of the PNA is labeled byfirst condensing a labeled moiety with the support upon which thelabeled PNA is to be assembled. Next, the first synthon of the PNA iscondensed with the labeled moiety. Alternatively, one or more spacermoieties can be introduced between the labeled moiety and the PNAoligomer (e.g. 8-amino-3,6-dioxaoctanoic acid). After the PNA iscompletely assembled and labeled, the PNA is cleaved from the support,deprotected and purified using standard methodologies.

[0065] For example, the labeled moiety could be a lysine derivativewherein the ε-amino group is labeled with a detectable moiety such as5(6)-carboxyfluorescein. Alternatively, the labeled moiety could be alysine derivative wherein, the ε-amino group is derivatized with a4-aminobenzoic acid moiety (e.g.4-(N-(tert-butyloxycarbonyl)-aminobenzamide). Condensation of the lysinederivative with the support would be accomplished using standardcondensation (peptide) chemistry. The α-amino group of the lysinederivative could then be deprotected and the PNA assembly initiated bycondensation of the first PNA synthon with the α-amino group of thelysine amino acid. After complete assembly, the PNA oligomer would thenbe cleaved from the support, deprotected and purified using well knownmethodologies.

[0066] Alternatively, a functional group on the assembled, or partiallyassembled, polymer is labeled with a donor or acceptor moiety (e.g. aPNA Molecular Beacon or a Linear Beacon) while it is still supportbound. This method requires that an appropriate protecting group beincorporated into the oligomer to thereby yield a reactive functional towhich the donor or acceptor moiety is linked but has the advantage thatthe label (e.g. a fluorophore) can be attached to any position withinthe polymer including within the probing nucleobase sequence. Forexample, the ε-amino group of a lysine could be protected with a4-methyl-triphenylmethyl (Mtt), a 4-methoxy-triphenylmethyl (MMT) or a4,4′-dimethoxytriphenylmethyl (DMT) protecting group. The Mtt, MMT orDMT groups can be removed from PNA (assembled using commerciallyavailable Fmoc PNA monomers and polystyrene support having a PAL linker;PerSeptive Biosystems, Inc., Framingham, Mass.) by treatment of theresin under mildly acidic conditions. Consequently, the donor oracceptor moiety can then be condensed with the ε-amino group of thelysine amino acid. After complete assembly and labeling, the polymer isthen cleaved from the support, deprotected and purified using well knownmethodologies.

[0067] Alternatively, a label (including one of the donor or acceptormoiety wherein the other of the donor or acceptor moiety is linked tothe PNA during assembly) is attached to the PNA after it is fullyassembled, cleaved from the support and optionally purified. This methodis preferable where the label is incompatible with the cleavage,deprotection or purification regimes commonly used to manufacture PNA.By this method, the PNA will generally be labeled in solution by thereaction of a functional group on the PNA and a functional group on thelabel. Those of ordinary skill in the art will recognize that thecomposition of the coupling solution will depend on the nature of PNAand the label. The solution may comprise organic solvent, water or anycombination thereof. Generally, the organic solvent will be a polarnon-nucleophilic solvent. Non-limiting examples of suitable organicsolvents include acetonitrile, tetrahydrofuran, dioxane andN,N′-dimethylformamide.

[0068] Generally the functional group on the PNA will be an amine andthe functional group on the label will be a carboxylic acid or activatedcarboxylic acid. Non-limiting examples of activated carboxylic acidfunctional groups include N-hydroxysuccinimidyl esters. If the label isan enzyme, preferably the amine on the PNA will be an arylamine. Inaqueous solutions, the carboxylic acid group of either of the PNA orlabel (depending on the nature of the components chosen) can beactivated with a water soluble carbodiimide. The reagent,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), is acommercially available reagent sold specifically for aqueous amideforming condensation reactions.

[0069] Generally, the pH of aqueous solutions will be modulated with abuffer during the condensation reaction. Preferably, the pH during thecondensation is in the range of 4-10. When an arylamine is condensedwith the carboxylic acid, preferably the pH is in the range of 4-7. Whenan alkylamine is condensed with a carboxylic acid, preferably the pH isin the range of 7-10. Generally, the basicity of non-aqueous reactionswill be modulated by the addition of non-nucleophilic organic bases.Non-limiting examples of suitable bases include N-methylmorpholine,triethylamine and N,N-diisopropylethylamine. Alternatively, the pH ismodulated using biological buffers such as(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium bicarbonate.

[0070] Exemplary Labels:

[0071] Numerous detectable moieties may be used for the practice of thisinvention. Suitable detectable or independently detectable moieties willbe chosen to be compatible with the assay to be performed. Generally,the labels will be chosen so that the label pair is neutral orpositively charged so that when labeled, the non-nucleotide probe doesnot exhibit a substantial affinity for the matrix. Alternatively, theprobe can be designed to incorporate charges (generally positivecharges) so that even if the net charge of the labels is negative, thelabeled probe is neutral or positively charged and therefore exhibitslittle or no affinity for the matrix.

[0072] Non-limiting examples of detectable moieties (labels) suitablefor use in the practice of this invention would include dextranconjugates, a branched nucleic acid detection system, chromophores,fluorochromes, spin labels, radioisotopes, mass labels, enzymes, haptensand chemiluminescent compounds. Preferred labeling reagents will besupplied as carboxylic acids or as the N-hydroxysuccinidyl esters ofcarboxylic acids. Numerous amine reactive labeling reagents arecommercially available (as for example from Molecular Probes, Eugene,Oreg.). Preferred fluorochromes (fluorophores) include5(6)-carboxyfluorescein (Flu),6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid (Cou), 5(and6)-carboxy-X-rhodamine (Rox), Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5)Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye,Cyanine 9 (Cy9) Dye (Cyanine dyes 3, 3.5, 5 and 5.5 are available as NHSesters from Amersham, Arlington Heights, Ill.) or the Alexa dye series(Molecular Probes). Preferred haptens include 5(6)-carboxyfluorescein,2,4-dinitrophenyl, digoxigenin, and biotin. Preferred enzymes includesoybean peroxidase, alkaline phosphatase and horseradish peroxidase.Other suitable labeling reagents and preferred methods of attachmentwould be recognized by those of ordinary skill in the art of PNAsynthesis.

[0073] Non-Nucleotide “Beacon” Probes:

[0074] The labels attached to the non-nucleotide “Beacon” probescomprise a set (hereinafter “Beacon Set(s)”) of energy transfer moietiescomprising at least one energy transfer donor and at least one energytransfer acceptor moiety. Typically, the Beacon Set will include asingle donor moiety and a single acceptor moiety. Nevertheless, a BeaconSet may contain more than one donor moiety and/or more than one acceptormoiety. The donor and acceptor moieties operate such that one or moreacceptor moieties accepts energy transferred from the one or more donormoieties or otherwise quench signal from the donor moiety or moieties.Though the previously listed fluorophores (with suitable spectralproperties) might also operate as energy transfer acceptors, preferably,the acceptor moiety is a quencher moiety. Preferably, the quenchermoiety is a non-fluorescent aromatic or heteroaromatic moiety. Thepreferred quencher moiety is 4-((-4-(dimethylamino)phenyl)azo) benzoicacid (dabcyl).

[0075] Transfer of energy between donor and acceptor moieties of anon-nucleotide “Beacon” probe may occur through collision of the closelyassociated moieties of a Beacon Set or through a nonradiative processsuch as fluorescence resonance energy transfer (FRET). For FRET tooccur, transfer of energy between donor and acceptor moieties of aBeacon Set requires that the moieties be close in space and that theemission spectrum of a donor(s) have substantial overlap with theabsorption spectrum of the acceptor(s) (See: Yaron et al. AnalyticalBiochemistry, 95: 228-235 (1979) and particularly page 232, col. 1through page 234, col. 1). Alternatively, collision mediated(radiationless) energy transfer may occur between very closelyassociated donor and acceptor moieties whether or not the emissionspectrum of a donor moiety(ies) has a substantial overlap with theabsorption spectrum of the acceptor moiety(ies) (See: Yaron et al.,Analytical Biochemistry, 95: 228-235 (1979) and particularly page 229,col. 1 through page 232, col. 1). This process is referred to asintramolecular collision since it is believed that quenching is causedby the direct contact of the donor and acceptor moieties (See: Yaron etal.).

[0076] (i) Linear Beacons:

[0077] In a preferred embodiment, the non-nucleotide “Beacon” probe is aLinear Beacon as more fully described in co-pending patent applicationU.S. Ser. No. 09/179,162 and WIPO publication WO99/22018, entitled“Methods, Kits And Compositions Pertaining To Linear Beacons”, hereinincorporated by reference.

[0078] (ii) PNA Molecular Beacons:

[0079] In a preferred embodiment, the non-nucleotide “Beacon” probe is aPNA Molecular Beacon as more fully described in co-pending patentapplication: U.S. Ser. No. 09/179,298 and WIPO publication WO99/21881,entitled “Methods, Kits And Compositions Pertaining To PNA MolecularBeacons”, herein incorporated by reference.

[0080] Detecting Energy Transfer:

[0081] Hybrid formation of a non-nucleotide “Beacon” probe with a targetsequence can be monitored by measuring at least one physical property ofat least one member of the Beacon Set which is detectably different whenthe hybridization complex is formed as compared with when thenon-nucleotide “Beacon” probe exists in the absence of target sequence.We refer to this phenomenon as the self-indicating property ofnon-nucleotide “Beacon” probes. This change in detectable signal shallresult from the change in efficiency of energy transfer between thedonor and acceptor which results from hybridization of thenon-nucleotide “Beacon” probes. Preferably, the means of detection willinvolve measuring fluorescence of a donor or acceptor fluorophore of aBeacon Set. Most preferably, the Beacon Set will comprise at least onedonor fluorophore and at least one acceptor quencher such that thefluorescence of the donor fluorophore is will be used to detect,identify or quantitate hybridization of the non-nucleotide probe to thetarget sequence.

[0082] Other Non-Nucleotide Self-Indicating Probes:

[0083] In another embodiment, the non-nucleotide probes of thisinvention are self-indicating probes of the type described in WIPOpatent application WO97/45539. The self-indicating non-nucleotide probesdescribed in WO97/45539 differ as compared with non-nucleotide “Beacon”probes primarily in that no quencher or acceptor moiety is present inthe probes of WO97/45539. Preferably the probes of WO97/45539, as usedin this invention, are appropriately labeled peptide nucleic acids.

[0084] Detectable and Independently Detectable Moieties/MultiplexAnalysis:

[0085] In preferred embodiments of this invention, a multiplexprobe-based hybridization assay is performed. In a multiplex assay,numerous conditions of interest are simultaneously examined. Multiplexanalysis relies on the ability to sort sample components or the dataassociated therewith, during or after the assay is completed. Inpreferred embodiments of the invention, distinct independentlydetectable moieties are used to label the different non-nucleotideprobes of a set. The ability to differentiate between and/or quantitateeach of the independently detectable moieties provides the means tomultiplex a hybridization assay because the data which correlates withthe hybridization of each of the distinctly (independently) labelednon-nucleotide probes to a target sequence can be correlated with thepresence, absence or quantity of the target sequence sought to bedetected in a sample. Consequently, the probe-based multiplex assays ofthis invention may be used to simultaneously detect the presence,absence or amount of each of two or more target sequences which may bepresent the same sample and in the same assay.

[0086] Spacer/Linker Moieties:

[0087] Generally, spacers are used to minimize the adverse effects thatbulky labeling reagents might have on hybridization properties ofprobes. Linkers typically induce flexibility and randomness into theprobe or otherwise link two or more nucleobase sequences of a probe.Preferred spacer/linker moieties for non-nucleotide probes used for thepractice of this invention consist of one or more aminoalkyl carboxylicacids (e.g. aminocaproic acid) the side chain of an amino acid (e.g. theside chain of lysine or ornithine) natural amino acids (e.g. glycine),aminooxyalkylacids (e.g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids(e.g. succinic acid) or alkyloxy diacids (e.g. diglycolic acid).Spacer/linker moieties may also incidentally or intentionally beconstructed to improve the water solubility of the probe. Thespacer/linker moieties may also be designed to enhance the solubility ofthe oligomer.

[0088] Preferably, a spacer/linker moiety comprises one or more linkedcompounds having the formula: —Y—(O_(m)—(CW₂)_(n))_(o)—Z—. The group Yhas the formula: a single bond, —(CW₂)_(p)—, —C(O)(CW₂)_(p)—,—C(S)(CW₂)_(p)— and —S(O₂)(CW₂)_(p). The group Z has the formula NH,NR², S or O. Each W is independently H, R², —OR², F, Cl, Br or I;wherein, each R² is independently selected from the group consisting of:—CX₃, —CX₂CX₃, —CX₂CX₂CX₃, —CX₂CX(CX₃)₂, and —C(CX₃)₃. Each X isindependently H, F, Cl, Br or I. Each m is independently 0 or 1. Each n,o and p are independently whole numbers from 0 to 10.

[0089] Linked Polymer:

[0090] A linked polymer comprises two or more nucleobase sequences whichare linked by a linker. The probes of this invention include linkedpolymers wherein the probing nucleobase sequence is linked to one ormore additional peptide nucleic acid, peptide or enzyme molecules.

[0091] Hybridization Conditions/Stringency:

[0092] Those of ordinary skill in the art of nucleic acid hybridizationwill recognize that factors commonly used to impose or controlstringency of hybridization include formamide concentration (or otherchemical denaturant reagent), salt concentration (i.e., ionic strength),hybridization temperature, detergent concentration, pH and the presenceor absence of chaotropes. Optimal stringency for a probe/targetcombination is often found by the well known technique of fixing severalof the aforementioned stringency factors and then determining the effectof varying a single stringency factor. The same stringency factors canbe modulated to thereby control the stringency of hybridization ofnon-nucleotide probes to target sequences, except that the hybridizationof a PNA is fairly independent of ionic strength. Ionic strength willnot likely be a substantial factor in the stringency of mostnon-nucleotide probes having a sufficiently neutral or positivelycharged backbone. Optimal stringency for an assay may be experimentallydetermined by examination of each stringency factor until the desireddegree of discrimination is achieved.

[0093] Suitable Hybridization Conditions:

[0094] Generally, the more closely related the background causingnucleic acid contaminates are to the target sequence, the more carefullystringency must be controlled. Blocking probes may also be used as ameans to improve discrimination beyond the limits possible by mereoptimization of stringency factors. Suitable hybridization conditionswill thus comprise conditions under which the desired degree ofdiscrimination is achieved such that an assay generates an accurate(within the tolerance desired for the assay) and reproducible result.Aided by no more than routine experimentation, those of skill in the artwill easily be able to determine appropriate hybridization conditionsfor performing an assay.

[0095] Blocking Probes:

[0096] Blocking probes are non-nucleic acid or nucleic acid probes whichcan be used to suppress the binding of the probing nucleobase sequenceof a probe to a hybridization site which is unrelated or closely relatedto the target sequence (See: Coull et al., PCT/US97/21845, a.k.a.WO98/24933). Generally, the blocking probes suppress the binding of theprobing nucleobase sequence to closely related non-target sequencesbecause the blocking probe hybridizes to the non-target sequence to forma more thermodynamically stable complex than is formed by hybridizationbetween the probing nucleobase sequence and the non-target sequence.Thus, blocking probes are typically unlabeled probes used in an assay tothereby suppress non-specific signal. Because they are usually designedto hybridize to closely related non-target sequence sequences, typicallya set of two or more blocking probes will be used in an assay to therebysuppress non-specific signal from non-target sequences which could bepresent and interfere with the performance of the assay.

[0097] Suitable Electrostatic Binding Conditions:

[0098] It is an important feature of the present invention that theelectrostatic binding conditions be chosen such that the non-nucleotideprobe exhibit little or no affinity for the matrix as compared withnucleic acid components of the sample. The electrostatic immobilizationof nucleic acids to matrices primarily involves the formation of saltpairs between the nucleic acid and the matrix. A salt pair comprises acharged species of the nucleic acid interacting with a counter chargedspecies of the matrix to form an interaction which tends to stabilizethe association of the nucleic acid to the matrix. Variable factorswhich will most affect electrostatic binding will involve modulation ofone or both the pH and/or ionic strength. The pH is an important factorsince it may affect the charge density on the matrix as well as the netcharge of the nucleic acid. Similarly, ionic strength will affect saltpair stability since it is well known in the chromatographic arts thatincreasing ionic strength will destabilize the interactions formedbetween nucleic acids and anion exchange stationary phases. For thepurposes of this invention, electrostatic binding conditions shall beconditions which allow for the reversible binding of the nucleic acid ofinterest to a matrix through salt pair formation.

[0099] Harmonization of Suitable Hybridization Conditions and SuitableElectrostatic Binding Conditions:

[0100] When employing the methods, kits and compositions of thisinvention, it is important to distinguish between suitable hybridizationconditions, wherein sequence specific hybridization of a non-nucleotideprobe to at target sequence is optimized, as compared with electrostaticbinding conditions which simply refer to conditions under which thenucleic acid binds to the matrix but the non-nucleotide probe or probesdo not exhibit a substantial affinity for the matrix. Typically, theelectrostatic binding conditions will be chosen such that thenon-nucleic probe is sufficiently neutral or positively charged. Becauseof the differences in the charges of the backbones of nucleic acid andnon-nucleotide probes of this invention, there is a broad range ofconditions within which the nucleic acid of interest binds to the matrixbut the non-nucleotide probe or probes do not. This principle isexemplified in Example 13 wherein it is clear that the non-nucleotideprobes do not interact with the matrix under any conditions examined butthe most nearly equivalent nucleic acid probes can interact with thematrix under many of the conditions tested whether or not the targetsequence is present in the sample.

[0101] Because it is an important feature of this invention that thenon-nucleotide probe hybridize to a nucleic acid which iselectrostatically bound to a matrix, it will be appreciated by one ofskill in the art that hybridization conditions (stringency factors) andelectrostatic binding conditions should be harmonized within the contextof the assay to be performed. Since pH and ionic strength are factors tobe considered in both stringency and electrostatic binding and since theelectrostatic binding conditions are broad as compared with optimizedstringency, it should always be possible to easily fix the electrostaticbinding conditions and then optimize probe discrimination by modulationof other stringency factors. In this respect, the methods of thisinvention is far superior to current methods known in the art (e.g.Arnold et al, U.S. Pat. No. 5,599,667). Aided by no more than routineexperimentation, those of skill in the art will easily be able toharmonize the electrostatic binding conditions and suitablehybridization conditions for performing an assay.

[0102] Matrices:

[0103] Generally, the matrix is merely a scaffold which is potentiallyseparable from the bulk fluid of the assay and which comprises chargedfunctional groups to which the nucleic acid reversibly bindselectrostatically. Typically, electrostatic binding occurs by salt pairformation between charged groups of the nucleic acid backbone andcharged groups of the matrix. For binding nucleic acids, the primaryinteractions will most likely involve formation of a salt pair betweenthe negatively charged phosphate groups of the nucleic acidphosphodiester backbone and positively charged functional groups of thematrix.

[0104] Non-limiting examples of suitable matrices include: polymerswhich are insoluble in water or mixtures of water and water solubleorganic solvents; two and three dimensional surfaces such as a wall of atube, a glass frit or a wafer; beaded supports such as magnetic beads,chromatographic packing supports, media and resins; porous beadedsupports such as chromatographic packing supports, media and resins(e.g. anion exchange chromatography media), a cast polymer such as amembrane (e.g. polyvinylidene difluoride, Teflon, polyethylene,polypropylene or polysulfone); co-polymeric materials and gels (e.g.polyacrylamide or agarose). In a preferred embodiment, commerciallyavailable ion exchange chromatographic media, and particularly thebeaded media, will be used as the matrix.

[0105] Matrix Shielding:

[0106] In certain preferred embodiments, the matrix may be temporarilyshielded from the assay components to thereby temporarily delay theelectrostatic binding of nucleic acid components of the assay to thematrix. For example, it may be preferable to partially or wholly shieldthe matrix from assay components until a nucleic acid synthesis oramplification reaction is partially or substantially completed so as tonot inhibit the synthesis or amplification (See for example: Example 12of this specification).

[0107] Those of skill in the art will recognize that partitioning ofreaction components may be obtained by preparing a reaction vesselhaving suitable compartments. Preferably however, the matrix will beshielded by the aid of a fluid which will serve as a temporary barrieruntil the reaction components are mixed. Thus, a preferable fluid willbe a water miscible fluid which is viscous and/or dense as compared withwater such that it does not readily blend with aqueous solutions untilit is subjected to heating or physical agitation. It will be appreciatedby those of skill in the art that a non-limiting example of such a fluidis glycerol.

[0108] Exemplary Assay Formats:

[0109] The methods, kits and compositions of this inventionsubstantially simplify the preparation and/or analysis of nucleic acidsof interest. It is also an advantage that they are generally applicableto all types of samples and assay formats typically used for theanalysis of nucleic acids. Several non-limiting examples of preferredassay formats which have been tested are described below. These examplesdemonstrate the broad applicability of the methods, kits andcompositions of this invention. Generally, the assay formats describedbelow are not necessarily mutually exclusive and one or more can becombined for the analysis of a particular sample.

[0110] (i) Amplification Assay Formats:

[0111] This invention is applicable to samples wherein the nucleic acidhas been synthesized or amplified. Non-limiting examples of preferrednucleic acid synthesis or nucleic acid amplification reactions wellknown in the art include Polymerase Chain Reaction (PCR), Ligase ChainReaction (LCR), Strand Displacement Amplification (SDA),Transcription-Mediated Amplification (TMA), Rolling Circle Amplification(RCA) and Q-beta replicase. When combined with non-nucleotide “Beacon”probes, these assay formats can be performed in a self-indicatingformat, including real-time as well as end-point determination whenusing a suitable instrument such as a Prism 7700 (PE Biosystems, FosterCity, Calif.). In a self-indicating assay, once the components of theassay have been combined, there is no need to disturb contents of theassay to determine the result. Since the assay contents need not bedisturbed to determine the result, there must be some detectable ormeasurable change which occurs and which can be observed or quantitatedwithout physically manipulating the contents of the assay. Manyself-indicating assays rely on a change in fluorescence which can beobserved with the eye or otherwise detected and/or quantitated with afluorescence instrument. Example 12 of this specification describesself-indicating PCR assays suitable for either real-time or end-pointanalysis.

[0112] (ii) Protection/Digestion Assays:

[0113] Other preferred assays of this invention are directed to thedetection of nucleic acids target sequences in samples; particularlywithin complex biological samples. Complex biological samples such asblood, urine, sputum and cell lysates typically require substantialprocessing in order to remove the bulk matter, such as protein, lipids,cellular debris, etc. which otherwise reduce the sensitivity and/orreliability of a probe-based hybridization assay. It has previously beendemonstrated that nucleic acid analogs, such as PNA, can hybridize to atarget sequence and thereby protect the nucleic acid target sequencefrom digestion/degradation by nucleases (See: Stanley et al.; U.S. Pat.No. 5,861,250). In this preferred assay format, one or more detectablenon-nucleic acid probes which can protect the nucleic acid target fromdigestion/degradation, are hybridized to the one or more targetsequences under suitable hybridization conditions. Preferably, thetarget sequence exists in a complex biological sample. After thenon-nucleic acid probe has been hybridized to the nucleic acid targetsequence, the sample is treated with one or more enzymes such asproteases and/or nucleases which degrade the sample components, possiblyincluding the nucleic acid molecule of interest but not thenon-nucleotide probe/target sequence complex. Because this treatmentdigests/degrades contaminating polymers and debris, this treatmentreduces or eliminates the sample complexity and/or processingrequirements normally associated with complex biological samples. Sincethe one or more non-nucleotide probe/target sequence hybrids, if presentin the sample, are intact after the enzyme treatment, they can beconcentrated on a matrix and detected as the means to detect, identifyor quantitate the target sequence in the sample of interest. An exampleof this assay format is found in Example 14 of this specification.Optionally, the detectable non-nucleotide probe/target sequence hybridis released from the matrix by adjustment of conditions outside therange required for electrostatic binding and thereby facilitatesdetection of the unbound non-nucleotide probe/target sequence hybrid, orjust the detectable probe, as the means to detect, identify orquantitate the target sequence in the sample.

[0114] Advantageously, the presence of the matrix is not required forprotecting the target sequence from degradation. Therefore, the matrixcan either be present during the enzyme treatment or added after theenzyme treatment to thereby electrostatically immobilize thenon-nucleotide probe/target sequence. In preferred embodiments, thenucleic acid analog is a non-nucleotide “Beacon” probe and the presence,absence or amount of self-indicating signal detected on the matrix isused to determine the presence, absence or amount of target sequencepresent in the sample or complex biological sample.

[0115] In a preferred embodiment of this assay, the assay temperature isadjusted and/or controlled so that imperfect hybrids are preferentiallydissociated in order to achieve a higher degree of target sequencediscrimination, including single point mutation discrimination.Generally, this involves adjusting the temperature of the assay to apoint below the melting temperature of the non-nucleotide probe/targetsequence hybrid so that non-nucleotide probe/non-target sequences are atleast partially dissociated. Since the non-nucleotide probe/non-targetsequence hybrids are generally less complementary as compared with thenon-nucleotide probe/target sequence hybrids, the optimal assaytemperature is typically within a fifteen degree range wherein thisrange is defined as five degrees above and ten degree below the meltingtemperature of the hybrid formed from the non-nucleotide probe and thenon-target sequence. For example, if the non-nucleotide probe/non-targetsequence sought to be discriminated in the assay has a meltingtemperature of 70° C., under assay conditions, the preferred range foradjusting the temperature of the assay would be between 75° C. (+5° C.)and 60° C. (−10° C.).

[0116] Dissociation of the non-nucleotide probe/non-target sequencesmakes the non-target sequences available for enzyme degradation sincethey are no longer protected. Though hybridization, particularly nearthe Tm, is an equilibrium process, destruction of the non-targetsequence prevents reassociation of the non-nucleotide probe/non-targetsequence hybrid and the generation of the non-specific signal associatedtherewith.

[0117] As previously stated, the assay formats described herein are notmutually exclusive to other assay formats. It is an important feature ofthis invention that the non-nucleotide probe/target sequence hybrid isreversibly bound to the matrix. Therefore the non-nucleotideprobe/target sequence hybrid can be released from the matrix forsubsequent analysis. Consequently, this Protection/Digestion Assay canalso be used merely for sample preparation or as a confirmatoryprecursor assay to a secondary assay. For example, theProtection/Digestion Assays can be combined with other assay formats,such as for the analysis of arrays or for sample preparation, beforeperforming a line assay or cytometric (flow or static) assay asdescribed below.

[0118] (iii) Line Assays:

[0119] Another preferred assay format useful for the practice of thisinvention is the line assay. A common line assay is a lateral flowassay. Many methods and devices for lateral flow assays are known (See:U.S. Pat. Nos. 5,916,521, 5,798,273, 5,770,460, 5,710,005, 5,415,994,4,956,302 and 4,943,522, all of which are herein incorporated). Aclassic example of a lateral flow assay is a commercially availablepregnancy test. In a classic pregnancy test, a sample of urine isapplied to a spot on a lateral flow assay device. The lateral flowdevice comprises a fluid conducting matrix, such as a filter ormembrane, which causes the fluid (e.g. urine) to passively flow(generally through capillary action) from the spot of application to theother end of the conducting matrix. Present within the lateral flowdevice (or otherwise added to the urine sample prior to application tothe device) is a detectable antibody to the HCG hormone; said HCGhormone being present in the urine sample only if the subject ispregnant. As the urine flows laterally within the device, theHCG/antibody complex forms as the components interact. Also within thedevice is a line (or other geometric shape) of a substance (usuallyanother antibody) to which the HCG/antibody complex will bind andthereby concentrate to produce a detectable signal.

[0120] Consequently, another embodiment of this invention contemplates aline or lateral flow assay for the detection of a nucleic acid targetsequence. In the line assay of this invention, a line, lines or othergeometric shape of matrix is spatially fixed on the device so that anynon-nucleic acid probe/target sequence complexes present in a sample canbe concentrated on the line (or other geometric shape) of the device asthe sample (or sample components) flow past. Preferably the assay is alateral flow assay. In the line assay of this invention, the detectablenon-nucleic acid probe can be added to the sample before or after thetarget sequence is concentrated on the matrix of the line device.Consequently, the target sequence in the sample is determined bydetecting, identifying or quantitating the non-nucleic acid probe/targetsequence complex as concentrated on the line, lines or other geometricshape. Example 16 of this specification is an example of a line assay.Optionally, the non-nucleotide probe/target sequence hybrid is releasedfrom the matrix by adjustment of conditions outside the range requiredfor electrostatic binding and thereby facilitates detection of theunbound non-nucleotide probe/target sequence hybrid, or just thedetectable probe, as the means to detect, identify or quantitate thetarget sequence in the sample.

[0121] (iv) Array Assay Formats:

[0122] In one embodiment, arrays are surfaces to which two or moresamples of interest have been immobilized, each at a unique location.Arrays comprising nucleic acid have been described in the literature. Itis an advantage of this invention that nucleic acid of a sample iseasily electrostatically immobilized to a surface under a broad range ofconditions. Therefore, an array of samples can be easily producedgenerally by just spotting (under electrostatic binding conditions) twoor more samples containing nucleic acid at unique locations on apositively charged surface. Because the location of each sample isknown, arrays of electrostatically immobilized nucleic acid cangenerally be used to simultaneously detect, identify or quantitate oneor more target sequences in two or more samples of interest. Thus, anarray of electrostatically immobilized nucleic acid may be useful indiagnostic applications or in screening compounds for leads which mightexhibit therapeutic utility. An example of an array assay is Example 15of this specification. Optionally, the non-nucleotide probe/targetsequence hybrid is released from the matrix by adjustment of conditionsoutside the range required for electrostatic binding and therebyfacilitates detection of the unbound non-nucleotide probe/targetsequence hybrid, or just the detectable probe, as the means to detect,identify or quantitate the target sequence in the sample.

[0123] It is an advantage of this invention that the non-nucleotideprobe is not necessary for the immobilization of the nucleic acid to thearray matrix. Therefore, the non-nucleotide probe may be added to theone or more samples before or after they are electrostaticallyimmobilized to the array matrix.

[0124] Arrays comprised of non-nucleotide probes/target sequence hybridshave the additional advantage that they are highly stable and should notbe degraded by enzymes which degrade nucleic acid. Therefore, thesearrays, or the samples which are to be applied to the array matrix, canbe treated as described above in the Section entitled“Digestion/Protection Assays” as a means to improve the assayperformance by the degradation of sample contaminates. Regardless ofwhether the sample is treated with enzyme before or after it is appliedto the array matrix, it is important that the non-nucleotideprobe/target sequence be formed so that the target sequence is protectedagainst degradation.

[0125] (v) Flow or Static Cytometric Assays:

[0126] Flow and static cytometry are very useful for the analysis ofwhole cells as well as particles. Because the matrices of this inventioncan be beaded or particulate, this invention is particularly well suitedfor the static or flow cytometric analysis of particles containingnucleic acids electrostatically immobilized thereto. According to thisinvention, the nucleic acid is electrostatically immobilized toparticles or beads. A non-nucleotide probe is used to detect a targetsequence of interest present on the particles or beads and can be addedbefore or after the nucleic acid is immobilized. Detection,identification or quantitation of the non-nucleotide probe/targetsequence complex in the static or flow cytometer is used as the means todetect, identify or quantitate the target sequence in the sample ofinterest. Example 13 of this specification utilizes a staticquantitation of fluorescence as the means to quantitate target sequenceelectrostatically immobilized to beads. Optionally, the non-nucleotideprobe/target sequence hybrid is released from the matrix by adjustmentof conditions outside the range required for electrostatic binding andthereby facilitates detection of the unbound non-nucleotide probe/targetsequence hybrid, or just the detectable probe, as the means to detect,identify or quantitate the target sequence in the sample.

[0127] Exemplary Applications For Using The Invention:

[0128] Because the methods, kits and compositions of this invention maybe used in a probe-based hybridization assay, this invention will findutility in improving assays used to detect, identify of quantitate thepresence or amount of an organism or virus in a sample through thedetection of target sequences associated with the organism or virus.(See: U.S. Pat. No. 5,641,631, entitled “Method for detecting,identifying and quantitating organisms and viruses” herein incorporatedby reference). Similarly, this invention will also find utility in anassay used in the detection, identification or quantitation of one ormore species of an organism in a sample (See U.S. Pat. No. 5,288,611,entitled “Method for detecting, identifying and quantitating organismsand viruses” herein incorporated by reference). This invention will alsofind utility in an assay used to determine the effect of antimicrobialagents on the growth of one or more microorganisms in a sample (See:U.S. Pat. No. 5,612,183, entitled “Method for determining the effect ofantimicrobial agents on growth using ribosomal nucleic acid subunitsubsequence specific probes” herein incorporated by reference). Thisinvention will also find utility in an assay used to determine thepresence or amount of a taxonomic group of organisms in a sample (See:U.S. Pat. No. 5,601,984, entitled “Method for detecting the presence ofamount of a taxonomic group of organisms using specific r-RNAsubsequences as probes” herein incorporated by reference).

[0129] The methods, kits and compositions of this invention areparticularly useful for the rapid, sensitive, reliable and versatiledetection of target sequences which are particular to organisms whichmight be found in food, beverages, water, pharmaceutical products,personal care products, dairy products or environmental samples. Theanalysis of preferred beverages include soda, bottled water, fruitjuice, beer, wine or liquor products. Consequently, the methods, kitsand compositions of this invention will be particularly useful for theanalysis of raw materials, equipment, products or processes used tomanufacture or store food, beverages, water, pharmaceutical products,personal care products, dairy products or environmental samples.

[0130] Likewise, the methods, kits and compositions of this inventionare particularly useful for the rapid, sensitive, reliable and versatiledetection of target sequences which are particular to organisms whichmight be found in clinical environments. Consequently, the methods, kitsand compositions of this invention will be particularly useful for theanalysis of clinical specimens or equipment, fixtures or products usedto treat humans or animals. For example, the assay may be used to detecta target sequence which is specific for a genetically-based disease oris specific for a predisposition to a genetically-based disease.Non-limiting examples of diseases include, β-Thalassemia, sickle cellanemia, Factor-V Leiden, cystic fibrosis and cancer related targets suchas p53, p10, BRC-1 and BRC-2.

[0131] II. Preferred Embodiments of the Invention:

[0132] Compositions:

[0133] In one embodiment, this invention pertains to compositionssuitable for detecting the presence of a target sequence in a sample. Apreferred composition comprises a nucleic acid, having a targetsequence, which is electrostatically bound to a matrix under suitableelectrostatic binding conditions. The composition further comprises anon-nucleotide probe having a probing nucleobase sequence which issequence specifically hybridized to at least a portion of the targetsequence provided however that the non-nucleotide probe does notsubstantially bind to the matrix under suitable electrostatic bindingconditions unless the target sequence is present on the matrix.Therefore, the electrostatic immobilization of the non-nucleotideprobe/target sequence complex to matrix is primarily determined by theinteraction of the nucleic acid with the matrix and not significantlydependent upon any interactions of the non-nucleotide probe and thematrix.

[0134] Such compositions are well suited for the detection of thepresence of target sequences in samples since the nucleic acidcomponents of the sample become concentrated on the matrix. Furthermore,the hybridized detectable non-nucleotide probe becomes concentrated onthe matrix such that the limit of detection of the assay can be improvedbecause the presence of the label is localized and thereby more easilydetected as compared with when it is distributed in bulk fluid (See:Example 12).

[0135] Methods:

[0136] In another embodiment, this invention pertains to methods for thedetection, identification or quantitation of a target sequence in asample containing nucleic acid. One exemplary method comprisescontacting a sample with a matrix and at least one non-nucleotide probewherein the nucleic acid in the sample will electrostatically bind tothe matrix under suitable electrostatic binding conditions.Additionally, the non-nucleotide probe will hybridize, under suitablehybridization conditions, to at least a portion of the target sequence,if present in the sample. The method further comprises detecting,identifying or quantitating the non-nucleotide probe/target sequencehybrid as a means to detect, identify or quantitate the target sequencein the sample.

[0137] In still another embodiment, this invention pertains to multiplexmethods for the detection, identification or quantitation of two or moretarget sequences of one or more nucleic acid molecules which may bepresent in the same sample. One exemplary method comprises contacting asample with a matrix and two or more independently detectablenon-nucleotide probes wherein the nucleic acid present in the samplewill electrostatically bind to the matrix under suitable electrostaticbinding conditions. Additionally, the two or more independentlydetectable non-nucleotide probes will hybridize, under suitablehybridization conditions, to at least a portion of the target sequenceswith which each probe is designed to hybridize if present in the nucleicacid of the sample. Consequently, if a particular target sequence iselectrostatically immobilized to the matrix, the independentlydetectable non-nucleotide probe designed to hybridized to thatparticular target sequence will become concentrated on the matrix and beavailable for detection. Therefore, the method further comprisesdetecting, identifying or quantitating each unique independentlydetectable non-nucleotide probe/target sequence hybrid which iselectrostatically bound to said matrix as a means to detect, identify orquantitate each unique target sequence sought to be detected in thesample and in the same assay. Optionally, the unique independentlydetectable non-nucleotide probe/target sequence hybrids is released fromthe matrix by adjustment of conditions outside the range required forelectrostatic binding and thereby facilitates detection of the unboundnon-nucleotide probe/target sequence hybrid, or just the detectableprobe, as the means to detect, identify or quantitate the targetsequence in the sample.

[0138] In still a further embodiment, this invention takes advantage ofthe stability of nucleic acid analog/nucleic acid complexes (See:Stanley et al.; U.S. Pat. No. 5,861,250) to thereby further improveassay performance and/or otherwise decrease the labor or complexity ofsample preparation. One exemplary method comprises contacting the samplewith at least one non-nucleotide probe wherein the non-nucleotide probewill hybridize, under suitable hybridization conditions, to at least aportion of the target sequence if present in the sample. The sample isalso contacted with a matrix wherein the nucleic acid molecule willelectrostatically bind to a matrix under suitable electrostatic bindingconditions. Either before or after immobilization to the matrix, thesample containing the non-nucleotide probe/target sequence complex iscontacted with one or more enzymes capable degrading samplecontaminates, including the nucleic acid molecule but not thenon-nucleotide probe/target sequence complex. The method furthercomprises detecting, identifying or quantitating the non-nucleotideprobe/target sequence hybrid as a means to detect, identify orquantitate the target sequence in the sample provided that thenon-nucleotide probe/target sequence is first immobilized to the matrix.Optionally, the detectable non-nucleotide probe/target sequence hybridis released from the matrix by adjustment of conditions outside therange required for electrostatic binding and thereby facilitatesdetection of the unbound non-nucleotide probe/target sequence hybrid, orjust the detectable probe, as the means to detect, identify orquantitate the target sequence in the sample.

[0139] In yet another embodiment, this invention relates to a method forthe detection, identification or quantitation of a target sequence of anucleic acid molecule electrostatically immobilized at a location on anarray wherein the array comprises nucleic acid moleculeselectrostatically bound at unique locations. One exemplary methodcomprises contacting the array with at least one non-nucleotide probe,wherein the non-nucleotide probe will hybridize, under suitablehybridization conditions, to at least a portion of the target sequenceif present on the array. The non-nucleotide probe/target sequencecomplex electrostatically bound at a location on said array is thendetected, identified or quantitated as the means to determine thepresence, absence or amount of target sequence present at said arraylocation. It is an advantage of the invention that one or more enzymescapable degrading sample contaminates, including the nucleic acid targetmolecule but not the non-nucleotide probe/target sequence complex, canalso be added before analysis of the array to thereby improve theperformance of the array assay by degrading sample contaminates whichmight otherwise lead to false positive results. Optionally, thedetectable non-nucleotide probe/target sequence hybrids can be releasedfrom the matrix by adjustment of conditions outside the range requiredfor electrostatic binding and thereby facilitates detection of theunbound non-nucleotide probe/target sequence hybrid, or just thedetectable probe, as the means to detect, identify or quantitate targetsequence in the sample. If the non-nucleotide probes are independentlydetectable, the analysis of the matrix can proceed in a multiplexformat.

[0140] In yet a further embodiment, this invention is directed to amethod for the detection, identification or quantitation of a targetsequence of a nucleic acid molecule which may be present in any of twoor more samples of interest. The method comprises mixing each of the twoor more samples of interest with at least one non-nucleotide probe,under suitable hybridization conditions. Next a matrix is contacted,under suitable electrostatic binding conditions, with at least a portionof each of the two or more samples to thereby electrostaticallyimmobilize the nucleic acid components of each sample to the matrix,each at a unique location, and thereby create a matrix array of samples.The non-nucleotide probe/target sequence complex electrostatically boundat a location on said array is then detected, identified or quantitatedas the means to determine the presence, absence or amount of targetsequence present at said array location. It is an advantage of theinvention that one or more enzymes capable degrading samplecontaminates, including the nucleic acid target molecule but not thenon-nucleotide probe/target sequence complex, can also be added beforeanalysis of the array to thereby improve the performance of the arrayassay by degrading sample contaminates which might otherwise lead tofalse positive results. Optionally, the detectable non-nucleotideprobe/target sequence hybrids can be released from the matrix byadjustment of conditions outside the range required for electrostaticbinding and thereby facilitates detection of the unbound non-nucleotideprobe/target sequence hybrid, or just the detectable probe, as the meansto detect, identify or quantitate target sequence in the sample. If thenon-nucleotide probes are independently detectable, the analysis of thematrix can proceed in a multiplex format.

[0141] Kits:

[0142] In yet another embodiment, this invention is directed to kitssuitable for performing an assay, as described herein, which detects thepresence, absence or number of target sequences in a sample. The kits ofthis invention comprise a matrix and one or more non-nucleotide probesand other reagents or compositions which are selected to perform anassay or otherwise simplify the performance of an assay used to detect,identify or quantitate a target sequence in a sample. Suitablenon-nucleotide probes, matrices and methods have been previouslydescribed herein. Typically, the kit will comprise a non-nucleotideprobe, a matrix and one or more reagents or buffers for fixing theelectrostatic binding conditions and/or hybridization conditions.

[0143] One embodiment of a preferred kit will comprise at least twoindependently detectable non-nucleotide probes such that the presenceabsence or amount of each independently detectable moiety can be used todistinctly identify or quantitate each of at least two target sequenceswhich may be present in a sample in the same assay (a multiplex assay).In a preferred embodiment, the kit will comprise two or morenon-nucleotide “Beacon” probes. Preferably, the kit will be useful forperforming a multiplex self-indicating assay such as a self-indicatingPCR assay.

[0144] Having described the preferred embodiments of the invention, itwill now become apparent to one of skill in the art that otherembodiments incorporating the concepts described herein may be used. Itis felt, therefore, that these embodiments should not be limited todisclosed embodiments but rather should be limited only by the spiritand scope of the following claims.

EXAMPLES

[0145] This invention is now illustrated by the following examples whichare not intended to be limiting in any way.

Example 1 Synthesis of DNA Oligonucleotides for Study

[0146] For this study, labeled and labeled DNA oligonucleotides suitableas probes or as nucleic acids comprising a target sequence were eithersynthesized using commercially available reagents and instrumentation orobtained from commercial vendors. All DNAs were obtained in purifiedform or purified using conventional methods. The sequences of the DNAoligonucleotides prepared are illustrated in Table 1, below. Methods andcompositions for the synthesis and purification of synthetic DNAs arewell known to those of ordinary skill in the art.

Example 2 Synthesis of N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OH

[0147] To 20 mmol of N-α-(Fmoc)-N-ε-(t-boc)-L-lysine-OH was added 60 mLof 2/1 dichloromethane (DCM)/trifluoroacetic acid (TFA). The solutionwas allowed to stir until the tert-butyloxycarbonyl (t-boc) group hadcompletely been removed from the N-α-(Fmoc)-N-ε-(t-boc)-L-lysine-OH. Thesolution was then evaporated to dryness and the residue redissolved in15 mL of DCM. An attempt was then made to precipitate the product bydropwise addition of the solution to 350 mL of ethyl ether. Because theproduct oiled out, the ethyl ether was decanted and the oil put underhigh vacuum to yield a white foam. The white foam was dissolved in 250mL of water and the solution was neutralized to pH 4 by addition ofsaturated sodium phosphate (dibasic). A white solid formed and wascollected by vacuum filtration. The product was dried in a vacuum ovenat 35-40° C. overnight. Yield 17.6 mmol, 88%.

Example 3 Synthesis of N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH

[0148] To 1 mmol of N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OH (Example 2) wasadded 5 mL of N,N′-dimethylformamide (DMF) and 1.1 mmol of TFA. Thissolution was allowed to stir until the amino acid had completelydissolved.

[0149] To 1.1 mmol of 4-((4-(dimethylamino)phenyl)azo)benzoic acid,succinimidyl ester (Dabcyl-NHS; Molecular Probes, P/N D-2245) was added4 mL of DMF and 5 mmol of diisopropylethylamine (DIEA). To this stirringsolution was added, dropwise, the N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OHsolution prepared as described above. The reaction was allowed to stirovernight and was then worked up.

[0150] The solvent was vacuum evaporated and the residue partitioned in50 mL of DCM and 50 mL of 10% aqueous citric acid. The layers wereseparated and the organic layer washed with aqueous sodium bicarbonateand again with 10% aqueous citric acid. The organic layer was then driedwith sodium sulfate, filtered and evaporated to an orange foam. The foamwas crystallized from acetonitrile (ACN) and the crystals collected byvacuum filtration. Yield 0.52 mmol, 52%.

Example 4 Synthesis of N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-PAL-Peg/PSSynthesis Support

[0151] The N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH (Example 2) was used toprepare a synthesis support useful for the preparation of C-terminaldabcylated PNAs. The fluorenylmethoxycarbonyl (Fmoc) group of 0.824 g ofcommercially available Fmoc-PAL-Peg-PS synthesis support (PerSeptiveBiosystems, Inc.; P/N GEN913384) was removed by treatment, in a flowthrough vessel, with 20% piperidine in DCM for 30 minutes. The supportwas then washed with DCM. Finally, the support was washed with DMF anddried with a flushing stream of argon.

[0152] A solution containing 0.302 gN-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH, 3.25 mL of DMF, 0.173 g[O-(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), 0.101 mL DIEA and 0.068 mL 2,6-lutidine wasprepared by sequential combination of the reagents. This solution wasthen added to the washed synthesis support and allowed to react for 2hours. The solution was then flushed through the vessel with a stream ofargon and the support washed sequentially with DMF, DCM and DMF. Theresin was then dried with a stream of argon.

[0153] The support was the treated with 5 mL of standard commerciallyavailable PNA capping reagent (PerSeptive Biosystems, Inc., P/NGEN063102). The capping reagent was then flushed from the vessel and thesupport was washed with DMF and DCM. The support was then dried with astream of argon. Finally, the synthesis support was dried under highvacuum.

[0154] Final loading of the support was determined by analysis of Fmocloading of three samples of approximately 6-8 mg. Analysis determinedthe loading to be approximately 0.145 mmol/g.

[0155] This synthesis support was packed into an empty PNA synthesiscolumn, as needed, and used to prepare PNA oligomers having a C-terminaldabcyl quenching moiety attached to the PNA oligomer through the ε-aminogroup of the C-terminal L-lysine amino acid.

Example 5 Synthesis of PNA

[0156] PNAs were synthesized using commercially available reagents andinstrumentation obtained from PerSeptive Biosystems, Inc. Doublecouplings were often performed to insure that the crude product was ofacceptable purity. Purity of the final PNAs was determined by standardreversed-phase chromatographic methods and the identity of the PNAconfirmed by comparison of theoretical calculated masses with results ofmass analysis using a MALDI-TOF mass spectrometer.

[0157] PNAs possessing a C-terminal dabcyl moiety were prepared byperforming the synthesis using the dabcyl-lysine modified synthesissupport prepared as described in Example 4. PNAs possessing anN-terminal fluorescein moiety were treated with the appropriate labelingreagents and linkers (as required) prior to cleavage from the synthesissupport (See: Example 6). Several methods are available for labeling aPNA oligomer with fluorescein but Applicants preferred method isdescribed in Example 6. PNAs comprising a Cy3 label (Amersham) werecleaved from the synthesis support and HPLC purified using conventionalmethods prior to Cy3 labeling as described in Example 7.

Example 6 Preferred Procedure for Labeling of Support Bound PNA with5(6)carboxyfluorescein

[0158] After proper reaction with linkers and removal of the terminalamine protecting group, the resin was treated with 250 μL of a solutioncontaining 0.5M 5(6)carboxyfluorescein, 0.5MN,N′-diisopropylcarbodiimide, 0.5M 1-hydroxy-7-azabenzotriazole (HOAt)in DMF (See: Weber et al., Bioorganic & Medicinal Chemistry Letters, 8:597-600 (1998). After treatment the synthesis support was washed anddried under high vacuum. The PNA oligomer was then cleaved, deprotectedand purified.

Example 7 General Procedure for Cy3 Labeling of PNAs

[0159] The purified amine containing PNA was dissolved in 1/1 DMF/waterat a concentration of approximately 0.05 OD/μL to prepare a stock PNAsolution. From the stock, approximately 30 nmole of PNA was added to atube. To this tube was then added 125 μL 0.1 M HEPES (pH 8.5), andenough 1/1 DMF/water to bring the total volume to 250 μL. This solutionwas thoroughly mixed. To a prepackaged tube of Cy3 dye (Amersham), wasadded the entire 250 μL solution prepared as described above. The tubewas well mixed and then allowed to react for 1 hour at ambienttemperature.

[0160] After reaction, the solvent was removed by evaporation in aspeed-vac. The pellet was then dissolved in 400 μL of a solutioncontaining 3:1 1% aqueous TFA/ACN. Optionally the solution was thentransferred to a 5000 MW Ultrafree (Millipore, P/N UFC3LCC25) or a 3000MW (Amicon, P/N 42404) and filtered to remove excess dye. The recoveredproduct was then repurified using conventional reversed phasechromatographic methods.

[0161] General Discussion of Examples 8 to 12:

[0162] The following Examples were performed to examine whether thepresence of target nucleic acids which had been electrostatically boundto polyethylene imine (PEI) derivatized beads could be specificallydetected using labeled PNA probes wherein the labeled (neutral) PNAwould not become immobilized to the beads in the absence of targetnucleic acid but would hybridize, and therefore become immobilized tothe beads, if the target nucleic acid was present. These experimentsdemonstrate an application which is uniquely suited to PNA probes sincethey possess a neutral backbone but nevertheless hybridize to nucleicacids with sequence specificity.

[0163] Aided by the discussion and examples described herein, those ofskill in the art will appreciate that positively charged matrices otherthan those coated with PEI are suitable for the practice of theinvention described herein. Similarly, those of skill in the art willappreciate that all kinds of matrices or supports, other than beads, aresuitable for the practice of this invention. Finally, those of skill inthe art will also appreciate that the non-nucleotide probes suitable forthe practice of this invention include present (e.g. PNA) and futureconstructs which sequence specifically interact with nucleic acid andwhich are either neutral or positively charged under conditions whereina nucleic acid will electrostatically bind to a charged matrix. TABLE 1Oligodeoxynucleotide Probes and Constructs Seq. ID DescriptionOligodeoxynucleotide Sequence No. KRASWT(24)Biotin-GTG-GTA-GTTA-GGA-GCT-GGT-GGC-GTA-OH 1 KRASMU(24)Biotin-GTG-GTA-GTT-GGA-GCT-TGT-GGC-GTA-OH 2 2 KRASMU(31)Biotin-GTG-GTA-GTT-GGA-GCT-TGT-GGC-GTA-GGC-AAG-A-OH 3 WT-15FluFlu-ACG-CCA-CCA-GCT-CCA-OH 4 MU-15Flu Flu-ACG-CCA-CAA-GCT-CCA-OH 5pBR322 5′ primer HO-GCT-TGT-TTC-GGC-GTG-GGT-AT-OH 6 pBR322 3′ primerHO-TAG-GTT-GAG-GCC-GTT-GAG-CA-OH 7 KRAS 5′ primerHO-ATG-ACT-GAA-TAT-AAA-CTT-GT-OH 8 KRAS 3′ primerHO-CTC-TAT-TGT-TGG-ATC-ATA-TT-OH 9 CompDNAHO-TCA-CTA-GTC-CCT-TCA-AGG-CTA-GCA-GTA-TAA- 10TGG-GTT-CTA-GGT-AAA-CGT-TCC-ACC-GTT-ACT-OH NonCompDNAHO-AGT-AAC-GGT-GGA-ACG-TTT-ACC-TAG-AAC-CCA- 11TTA-TAC-TGC-TAG-CCT-TGA-AGG-GAC-TAG-TGA-OH

[0164] All oligodeoxynucleotides are illustrated from the 5′ to the 3′.Stock solutions of the oligodeoxynucleotides were generally prepared bydissolving the dry powder in TE Buffer (TE Buffer: 10 mM TRIS pH 8.3, 1mM EDTA).

[0165] Materials: PEI-Silica beads (gel) Amicon P/N PAE-300-15HP-Sepharose Q beads Pharmacia Biotech P/N 17-1014-03

[0166] Commercially available PEI derivatized beads were chosen forthese experiments since they were readily available as wellcharacterized commercial anion exchange chromatography packing materialshaving a high density of positively charged functional groups per unitarea at neutral pH (pH of 7). Because they possessed a high density ofpositively charged functional groups at neutral pH, they possessed afavorable binding capacity for oligonucleotides which are negativelycharged (because each phosphodiester comprises a single negative chargethe charge of each nucleic acid is length dependent) at neutral pH.

[0167] PEI-Silica and PEI-Sepharose beads were determined to beessentially interchangeable in all experiments performed. However, thePEI-Silica beads were found to have an intrinsic (native) fluorescencewhen illuminated on the UV transilluminator, and for that reason may beless suitable in some applications. TABLE 2 PNA Probes Description PNASequence WT-15Flu Flu-OO-ACG-CCA-CCA-GCT-CCA-NH₂ MU-15FluFlu-OO-ACG-CCA-CAA-GCT-CCA-NH₂ MU-15 H₂N-OO-ACG-CCA-CAA-GCT-CCA-NH₂Blocker BK.RAS-Cy3 Cy3-O-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ UQ-Cy3Ce-OO-TGA-TTG-CGA-ATG-K(Cy3)-NH₂ RASWT-Cy3Cy3-OOE-ACG-CCA-CCA-GCT-CCA-E-NH₂ BioP-15Ac-OE-TTA-TAC-TGC-TAG-CCT-EO-K(Bio)-NH₂

[0168] All PNA sequences are written from the amine to the carboxylterminus. Abbreviations are: Ac=acetyl; Flu=5(6)-carboxyfluorescein;dabcyl=4-((4-(dimethylamino) phenyl)azo)benzoic acid; Bio=biotin;O=8-amino-3,6-dioxaoctanoic acid; K=the amino acid L-Lysine; E=thesolubility enhancer “4” as represented in Gildea et al., Tett. Lett. 39(1998) 7255-7258; Ce=the group obtained by capping the PNA with thecharged moiety “7” as represented in Gildea et al., Tett. Lett. 39(1998) 7255-7258 and Cy3=the cyanine 3 dye from Amersham. Stocksolutions of PNAs are generally prepared by dissolving the purifiedprobe in a solution containing 1/1 N,N′-dimethylformamide (DMF)/water ata concentration of approximately 0.05 OD (260 nm) per μL.

[0169] DNA Plasmid Templates:

[0170] The plasmids, pKRASMU(31) and pKRASWT, were used as templates inPCR amplification reactions and were generated by cloning a PCR ampliconfrom human DNA into the pCR2.1 plasmid (Invitrogen). The mutant humanDNA was prepared from a cell line, Calu-1, which contains a pointmutation at base 129 of the K-ras gene. The wild type human DNA wasprepared from a cell line, NCI, which contains two copies of the wildtype K-ras gene. Clones were screened by restriction fragment analysisand sequence analysis. Large preparations of the plasmid were generatedand quantitated using standard techniques. The amplified region flanksthe K-ras mutation and was 111 bp in length. dsDNA Template (amplifiedregion only) ¢3′primer hyb. site→ 3′GAGATAACAACCTAGTATAAGCAGGTGTTTTACTAAGACTTA . . . 5′CTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAAT . . .

[0171]                                 ¢Linear Beacon Hyb. site→ . . .ATCGACTTAGCAGTTCCGTGAGAACGGATGCGGTG (G/T) TC GAGGTT . . . . . .TAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC (C/A) AG CTCCAA . . .

[0172] . . . GATGGTGTTCAAATATAAGTCAGTA 5′ Seq. ID No. 12 (wt), 13 (mu)

[0173] . . . CTACCACAAGTTTATATTCAGTCAT 3′ Seq. ID No. 14, (wt), 15 (mu)

[0174] <-5′ primer hyb. site->

[0175] The position and sequence of the point mutation of the ampliconsis illustrated in parenthesis. Plasmid pBR322 was obtained from NewEngland BioLabs; P/N 300-3S. TABLE 3 Salt Buffers: Buffer ID BufferComponents: A 0 mM NaCl, 10 mM TRIS-Cl pH 8.0, 1 mM EDTA, 0.1% Tween-20.B 100 mM NaCl, 10 mM TRIS-Cl pH 8.0, 1 mM EDTA, 0.1% Tween-20. C 200 mMNaCl, 10 mM TRIS-Cl pH 8.0, 1 mM EDTA, 0.1% Tween-20. D 300 mM NaCl, 10mM TRIS-Cl pH 8.0, 1 mM EDTA, 0.1% Tween-20. E 400 mM NaCl, 10 mMTRIS-Cl pH 8.0, 1 mM EDTA, 0.1% Tween-20. F 500 mM NaCl, 10 mM TRIS-ClpH 8.0, 1 mM EDTA, 0.1% Tween-20.

[0176] Electrophoresis Supplies: 10-20% polyacrylamide gel: ESA, catalog# 80-0015 10 × Gel Buffer: ESA, catalog # 80-0132 4 × loading dye: 50%glycerol, 0.1 M Bromphenol Blue, 0.01 M Xylene Cyanol, 4 × ESA GelBuffer (diluted from ESA# 80-0132)

Example 8 General Properties of Polyethylene imine (PEI) Beads

[0177] Polyethylene imine (PEI) beads were examined to determinephysical characteristics such as particle size and shape as well aschemical properties such as binding capacity of nucleic acid. Particlesize and concentration of beads suspended per unit of volume weredetermined using a hemacytometer (Hausser Scientific; Horsham, Pa.;Model # 3900).

[0178] Using the hemacytometer, the Sepharose beads were found togenerally be spherical in shape and have a diameter in the range ofapproximately 20 to 50 μm with an estimated average diameter of 30 μm.The concentration of the suspended Sepharose beads was estimated to beapproximately 50,000 beads/μL, after being washed (per manufacturesinstructions) and redissolved in deionized water.

[0179] Using the hemacytometer, the silica beads were found to alsogenerally be spherical in shape and have a diameter in the range ofapproximately 5 to 15 μm with an estimated average diameter ofapproximately 10 μm. The concentration of the suspended silica beads wasestimated to be approximately 150,000-200,000 beads/μL, after beingwashed (per manufactures instructions) and redissolved in deionizedwater.

[0180] The capacity of the silica and Sepharose beads to bind nucleicacid was examined in 100 mM TRIS-HCl pH 7.5. The capacity of theSepharose beads was approximated to be about 0.75 OD₂₆₀ of nucleic acidper μL of beads, or approximately 1.1 E-5 OD₂₆₀ of nucleic acid perbead. The capacity of the silica beads was approximated by applicant tobe about 0.4 OD₂₆₀ of nucleic acid per μL of beads, or approximately 0.8E-6 OD₂₆₀ of nucleic acid per bead. For all experiments conducted, thesebinding capacities were high enough such that all the nucleic acid ofthe sample was expected to be electrostatically bound to the supportgiven the amount of matrix present in the assay and the electrostaticbinding conditions used.

Example 9 Preliminary Studies on [Salt] and pH

[0181] As discussed above, the silica and Sepharose beads arecommercially available anion exchange chromatography media. As anionexchange chromatography often utilizes a salt gradient or pH gradient toelute materials which are electrostatically bound thereto, the effect ofmodulation in salt concentration and pH variation on the binding of PNAand/or DNA probes to Sepharose beads was studied to determine suitableelectrostatic binding conditions for subsequent experimentation.

[0182] i. [Salt]

[0183] To individual tubes containing 5 μL of Sepharose beads and 94 μLof one of each of six salt solutions (Salt Buffers A-F, See: Table 3,above) was added 5 pmole (in 1 μL of an appropriate solvent) of eitherDNA WT-15Flu (Table 1) or PNA WT-15Flu (Table 2) probe. The tubes werevortexed, briefly centrifuged and then examined under UV light todetermine whether the fluorescently labeled probes were stillpredominately in solution or whether they had adsorbed onto the surfaceof the beads.

[0184] The PNA probe (PNA WT-15Flu, Table 2) was found to bepredominately adsorbed to the beads when in Salt Buffers A and B, butpredominately in solution when all the buffers of higher saltconcentration were used. This results indicated that the fluoresceinlabeled PNA probe would only bind to the beads at low ionic strength(approximately 200 mM NaCl or below).

[0185] By comparison, the fluorescein labeled DNA probe, (DNA WT-15Flu,Table 1) of identical subunit length and nucleobase sequence, was foundto be substantially adsorbed to the beads except when Buffer F waspresent. In Buffer F, some of the probe was observed in the solution.This data indicated that at least 500 mM NaCl was required to disruptthe electrostatic interactions of the 15-mer DNA probe and the PEI onthe surface to thereby free the probe from the matrix.

[0186] Though the difference of 200 mM NaCl to release the PNA 15mer ascompared with 500 mM for release of the DNA 15mer was significant enoughto be useful for the practice of may embodiments of this invention, therequirement for 200 mM NaCl to release PNA probe from the PEI surfacewas surprising and therefore prompted subsequent examination. Uponfurther analysis, the presence of the fluorescein label(5(6)-carboxyfluorescein), which possess two negative charges at neutralpH, was determined to be the primary reason for the strong electrostaticinteractions between the PNA probe and the PEI derivatized beads.

[0187] By way of example, a Cy3-labeled PNA 15-mer (UQ-Cy3, Table 2)which comprised a positively charged capping group was examined in theSalt Buffers listed in Table 3, above. It was found that the Cy3 labeledPNA did not bind to the beads even in Salt Buffer A. Furthermore, freefluorescein was found to be substantially adsorbed to the beads in SaltBuffer A, whereas; the Cy3 dye did not. Finally, it was determined that1 μL of the Sepharose beads could adsorb as much as 5 pmole of5(6)-carboxyfluorescein when in Salt Buffer A. Consequently, this datasuggests that it was the fluorescein label and not the PNA portion ofthe oligomer which exhibited the strong interaction with the PEIderivatized beads in Salt Buffers A and B. Thus, it is believed thatnative PNA does not substantially electrostatically bind to the PEI evenunder low salt conditions (e.g. Salt Buffer A). This is consistent withexpectations since PNA is neutral and should therefore not be expectedto electrostatically bind to the PEI derivatized beads.

[0188] ii. pH Effects:

[0189] Because the net charge on the PNA backbone should not changedramatically at pH in the range of 5-10, the effect of modulation in pHupon the binding of PNA to PEI derivatized beads was not examined.However, the pH dependency of DNA binding to PEI derivatized silicabeads was examined to determine working parameters for subsequentexperimentation. The results of experiments can be summarized asfollows: At pH 7.3, the WT-15Flu DNA probe stayed bound to the silicabeads up to 0.8 M NaCl, whereas; at pH 8.3, the same probe did not bindto the beads at NaCl concentrations in excess of 0.1 M.

[0190] These results can be correlated with the number of expectedpositively charged functional groups of the PEI support. The highercapacity at pH 7.3 indicates that the support is more highly charged(higher charge density) under these conditions. However, at pH 8.3, thepH of the solution is approaching the pK of the secondary amine of thePEI and therefore the support begins to become neutralized (fewerpositive charges per unit area). With fewer positive charges, each beadhas a lower affinity for the negatively charged oligodeoxynucleotides.Therefore, less salt is required to disrupt the electrostaticinteractions and thereby release the DNA into the solution.

Example 10 Preliminary Examination of Hybrid Formation of ImmobilizedDNA

[0191] A study was conducted to determine whether hybridization of thePNA probes would occur with nucleic acid electrostatically immobilizedto the surface of the Sepharose beads. For this experiment, 1 pmole ofthe MU-15Flu PNA probe was allowed to hybridize to 0.5 pmole of theKRASMU(31) DNA target which was either free in solution orelectrostatically bound to Sepharose beads (1 μL of a 1:10 dilution ofbead stock in Buffer E). “PNA only” (probe) and “DNA only” (targetsequence) controls were also examined. The hybridization reactions wereallowed to proceed for approximately 2 minutes, after which, 1 μL of the1:10 dilution of bead stock was added to the sample which did notinitially contain Sepharose beads.

[0192] The hybridization reaction contents were then suctioned intoindividual capillary pipettes from which the liquid was wicked therebyleaving behind the beads and any bound and fluorescently labeled PNA/DNAhybrids. The “PNA only” and “DNA only” controls were found to benon-fluorescent under UV light. However, by visual inspection, both ofthe PNA/DNA hybridization reactions were equally fluorescent.Consequently, this data indicates that the PNA can hybridize withroughly equivalent efficiency to both the DNA electrostaticallyimmobilized to a Sepharose beads as well as it can hybridize to the DNAfree in solution.

Example 11 Efficiency of Electrostatic Capture and Release

[0193] A study was performed to determine the efficiency of theelectrostatic capture and release of nucleic acid at very low nucleicacid concentrations. Because the amount of nucleic acid was extremelysmall, the polymerase chain reaction (PCR) was used to quantify thecaptured and recovered nucleic acid. The plasmid pBR322 (New EnglandBiolabs; PN/P/N 300-3S) was used at concentrations ranging from 5 E+11to 5 E+5 molecules (approximately 1 attomole) per microliter.

[0194] Plasmid DNA was captured over a period of 5 minutes using 1 μL ofPEI-Silica beads in 20 μL of 100 mM TRIS-HCl, pH 7.6. After capture, thesamples were pelleted by centrifugation, the supernatants were removed,and the beads were washed with 1000 μL of 100 mM TRIS-HCl pH 7.6 toremoved non-specifically bound material. The plasmid DNA was thenreleased from the beads by treatment with 10 μL of a high salt buffercontaining 2 M NaCl and 100 mM TRIS-HCl pH 7.6. One microliter of eachbead eluate was then added to 99 μL of 100 mM TRIS-HCl pH 7.6 to dilutethe NaCl concentration down to a level acceptable for PCR. Twomicroliters of each diluted sample was then added to a 50 μL PCRreaction.

[0195] In addition, each PCR reaction also contained, 5 pmole of 5′pBR322 primer, 5 pmole of 3′ pBR322 primer, 3 mM MgCl₂, 250 μM NTPs, 2.0units AmpliTaq DNA polymerase, 50 mM KCl, and 10 mM Tris-HCl pH 8.3 (PCRreagents including 10× buffer, magnesium chloride solution, AmpliTaq DNApolymerase, and nucleotide triphosphates were obtained fromPerkin-Elmer, Foster City, Calif.). Reactions were performed inmini-eppendorf tubes using a Perkin-Elmer 2400 thermocycler. The PCRprotocol involved a 20 second warm up to 95° C. (1st round only),followed by denaturing at 95° C. for 20 seconds, annealing at 56° C. for20 seconds, and extension at 74° C. for 20 seconds. Thedenaturation-annealing-extension cycle was repeated for 30 cycles,followed by a final extension step at 74° C. for 5 minutes.

[0196] All of the samples were run on a polyacrylamide gel after PCR,and all contained detectable levels of the correct sized amplicon,though the most dilute sample (5 E+5 input molecules) was barelydetectable. The amount of DNA in the PCR reaction was actually 500 foldless (1 E+3 molecules) due the dilution necessary to remove salt, asdescribed above. In a control experiment run at the same time, 1 E+3molecules was the limit of detection of pBR322 by 30 cycles of PCR.These results suggest that at 5 E+5 molecules of input template, themajority of the plasmid DNA initially added to the matrix was capturedand released.

Example 12 Self-indicating PCR Assays

[0197] Self-indicating and closed-tube assays are becoming increasinglypopular for their ability to streamline, simplify and potentiallyautomate routine nucleic acid analysis assays. Also, closed-tube assaysprevent carry-over contamination between samples which is a major sourceof false positive results in nucleic acid diagnostics. Since theconcentration of detectable moieties is an advantage associated with theelectrostatic binding of nucleic acids to matrices, it was envisionedthat it might be possible to create a self-indicating PCR assay whereinthe fluorescence of the matrix enclosed in the reaction, at the time thereaction components are mixed, could be used to determine the result ofa PCR amplification by mere visual or instrument monitoring of the tubeduring and/or after PCR was completed.

[0198] Point mutation analysis is another important objective of anucleic acid diagnostic test since accurate determination of a specificpoint mutation of genetic material in a sample is often a decisivefactor in the proper identification of genetic disorders and otherdisease states. As discussed in the specification, blocker probes can beused to improve single point mutation analysis of a probe-based assaybeyond the limits which are possible by the precise optimization orcontrol of stringency. Thus, it was envisioned that the use of PNABlocker probes in conjunction with Linear Beacons would facilitate thedevelopment of a self-indicating probe based assay capable of pointmutation discrimination which would generate a result which could beinterpreted by visual inspection or by instrument analysis during and/orafter the PCR was completed.

[0199] For Examples A and B, asymmetric PCR was utilized becauseasymmetric PCR yields a significant excess of single stranded nucleicacid. Since it is possible to choose which of the strands of theamplicon are preferentially amplified by judicious adjustment of theratio of 5′ and 3′ primers, it was possible to design the assay so thatthe target sequence to which the non-nucleotide probe hybridizes wascontained within the over produced single stranded nucleic acid of theasymmetric PCR assay.

[0200] For Examples A and B, a Linear Beacon was chosen as thenon-nucleotide probe since Linear Beacons are inherently non-fluorescent(or very slightly fluorescent) until hybridized to the target sequence.This approach was advantageous since the reaction cocktail containingthe Linear Beacon would remain relatively non-fluorescent throughout theassay and in theory, only the beads would become fluorescent providedthe target sequence was generated and electrostatically bound to thematrix (beads). Thus, the Linear Beacon (BK.RAS-Cy3; See Table 2, above)was designed to hybridize to the over produced strand of a region ofdsDNA (See: illustration on p. 28) sought to be amplified and was addedto the PCR cocktail before thermocycling. The Linear PNA Beacon waslabeled with Cy3 since prior experiments had demonstrated that thenegatively charged fluorescein label exhibited an affinity for the PEIcoated beads at salt concentrations of less than 200 mM.

[0201] Though Linear Beacons may hybridize to the target sequence duringthermocycling, significant inhibition of the amplification process wasnot observed. Consequently, the PCR amplification was successfullymonitored using the detectable fluorescent signal of the Linear Beaconwhich was generated on the surface of the beads in response to theactivity of the PCR reaction. The data presented conclusivelydemonstrates the feasibility of using Linear Beacons for the detectionor point mutation analysis of nucleic acid electrostatically bound to amatrix which has been generated by amplification in a closed tube assay.As evidenced by FIGS. 1 and 3, the result can be determined by merevisual inspection of the final assay sample still in the tube. Sincefluorescence is visible to the naked eye, a sensitive instrument, suchas a Prism 7700, would be suitable for real-time or end-point automatedsample analysis. The figures further demonstrate that concentration ofthe samples on the matrix makes it possible to improve the limits ofdetection of the assay since the signal intensity on the beads is farmore intense than the signal generated by the bulk fluid when the LinearBeacon is free in solution.

[0202] A. Asymmetric PCR with Linear Beacons

[0203] PCR Materials & Methods:

[0204] This experiment comprised five individual PCR reactions. Variablefactors examined within the set of five reactions included the presenceor absence of PEI derivatized Sepharose beads (approximately 25,000PEI-Sepharose beads), the presence or absence of plasmid template(pKRASWT at 20 fmole per 50 μL reaction (0.4 nM)) and the presence orabsence of thermocycling (TMC). Table 4, below, summarizes thecomposition of various tubes with respect to these variable factors. Inaddition, each PCR reaction contained 1.5 μL of 100% glycerol, 0.5 μL ofwater (control) or Sepharose beads, 45 pmole of KRAS 5′ primer, 5 pmoleof the KRAS 3′ primers, 3 mM MgCl₂, 250 μM NTPs, 2.0 units AmpliTaq DNApolymerase, 50 mM KCl, 10 mM TRIS-Cl pH 8.3 and 50 pmole BK.RAS-Cy3non-nucleotide probe (Linear Beacon) in a total volume of 50 μL. Duringpreparation and prior to PCR, the tubes were carefully handled to avoidmixing of components.

[0205] The PCR protocol involved a 20 second warm up to 95° C. (1stround only), followed by denaturing at 95°C. for 5 seconds, annealing at55° C. for 30 seconds, and extension at 74° C. for 30 seconds. Thedenaturation-annealing-extension cycle was repeated for 50 cycles,followed by a final extension step at 74° C. for 5 minutes.

[0206] After thermocycling, the tubes were placed on a transilluminatorand the fluorescence examined by eye. Thereafter, the tubes werevortexed and then centrifuged for 2 minutes to concentrate the beads atthe tube bottom. No significant difference was observed whether or notthe tubes were vortexed and centrifuged before viewing. This indicatedthat the glycerol did not affect the end point result.

[0207] Notes:

[0208] 1. The glycerol was added to temporarily shield the beads fromthe reaction components in the early stages of PCR so that the processwould not be substantially inhibited by electrostatic binding of theprimers to the matrix (beads) during the critical early thermocycles.Subsequent investigations have demonstrated that the presence of atemporary shield is not essential to achieve an accurate result but isnevertheless preferred. TABLE 4 Variable Factors Tube # PEI-BeadsTemplate TMC 1 — pKRASWT no 2 — — yes 3 — pKRASWT yes 4 Sepharose — yes5 Sepharose pKRASWT yes

[0209] Post PCR Workup/Analysis:

[0210] After PCR, tubes 1-5 were placed on a transilluminator tovisualize fluorescence and then photographed. FIG. 1A is a negative ofthe scanned image of the photograph taken of the five mini-eppendorftubes immediately after PCR (tubes are labeled 1-5). After thephotograph was taken, 0.5 μL of the PEI-Sepharose bead stock was addedto tubes 1-3. The five tubes were then vortexed, centrifuged and againplaced on the transilluminator and again photographed. FIG. 1B is anegative of the scanned image of the second photograph of the fivemini-eppendorf tubes.

[0211] After re-photographing the tubes, the supernatants were decantedand the beads were washed with 100 μL of a solution containing 50 mMNaCl and 100 mM TRIS-HCl pH 7.6. Washing involved adding the washbuffer, vortexing briefly, centrifuging and then decanting. Theelectrostatically bound nucleic acids were then released from the beadsfor analysis by vortexing in a solution containing 10 μL of 0.05%ammonium hydroxide and 2.0 M NaCl. Supernatants were removed andtransferred to a microwell plate where they were neutralized with 1 μL0.1 N hydrochloric acid. To each well was added 4 μL of 4× loading dye.Finally, 15 μL of each sample was then run on a 10-20% gradient gel toconfirm nucleic acid amplification and identify product size.

[0212]FIGS. 2A and 2B are the negative of images of photographs of thesame 10-20% gradient polyacrylamide gel illuminated on a UVtransilluminator which were taken before and after ethidium bromidestaining, respectively.

[0213] Results:

[0214] Tube Images/Photographs

[0215] With regard to analysis of the images of the photographs, pleasenote that although black and white photographs were taken, visualinspection of the tubes was consistent with the dark to light contrastseen in the images except that the Cy3 dye appeared as orange to the eyeunder the transilluminator. Furthermore, the negative (generatedelectronically) of the scanned image is shown since it is believed thatthe contrasts of the negative image are superior for illustration andwill be more accurately reproduced by photocopying.

[0216] With reference to FIG. 1A, the results are as expected. Tube 1was a control which was not exposed to thermocycling and thereforelittle or no fluorescence was observed (the tube contents appear clearin contrast to the background). Likewise, tubes 2 and 4 contained notemplate and therefore little or no fluorescence was observed insolution (tube 2) or on the beads (tube 4) since no amplification shouldhave occurred. Tube 3, however, was fluorescent as expected sinceamplification of the template should have produced the 111 bp ampliconto which the Linear Beacon hybridized to generate detectable signal.Nevertheless, since no Sepharose was present, the solution was visiblyorange as compared with tubes 1, 2 and 4. Likewise, tube 5 containedhighly fluorescent (orange by eye; dark in the negative of the image)beads as expected since amplification of the template should haveproduced the 111 bp amplicon (electrostatically bound to the beadmatrix) to which the Linear Beacon hybridized to generate a detectablesignal.

[0217] With reference to FIG. 1B, the results are also as expected.Specifically, the addition of the Sepharose beads to tubes 1-3 onlyaffected the fluorescence of tube 3. In particular, the orangefluorescence which was observed to be in solution prior to the additionof the Sepharose beads, was concentrated on the bead surface (dark inthe negative of the image) after bead addition. Furthermore, theintensity of the fluorescence of tube 3, which could be determinedvisually, was comparable to the fluorescence intensity of the beads intube 5. Thus, there appears to be no difference in the result whether ornot the matrix is added before or after the PCR reaction is performed.

[0218] In summary, the data presented in FIGS. 1A and 1B indicate thatit is possible to perform self-indicating amplification assays whereinsignal of a probe can be concentrated on a matrix to which an amplifiedtarget nucleic acid is electrostatically immobilized.

[0219] Gel Photographs/Images:

[0220] Analysis of the PCR reactions by gel was performed to determinethe presence and size of amplicons to thereby confirm that the visualanalysis of the tubes correlated with expected products of PCRamplification.

[0221] With reference to FIGS. 2A and 2B, the wells of the gel are atthe top of the photographs. Aliquots of each tube were added near thetop of the gel and electrophoretically directed towards the bottom ofthe gel. Lanes 2 and 8 contain two different double stranded DNA sizemarkers; lane 2 is ØX174/HaeIII (New England BioLabs #303-1S) and lane 8is a 100 bp ladder (New England BioLabs #323-1L). Band sizes areindicated in FIG. 2B. Lanes 3-6 contain samples of released nucleic acidisolated from the beads in tubes 2 through 5 respectively and lane 7contains material released from the beads in tube 1.

[0222] With reference to FIG. 2A, the presence of inherently fluorescentbands can be seen in lanes 4 and 6 (from tubes 3 and 5 respectively)toward the bottom of the image. The fluorescent bands can be attributedto the presence of the Linear Beacon still hybridized to the nucleicacid amplicon even after it has migrated into the gel. This result isconsistent with amplification in tubes 3 and 5 as indicated by thevisual analysis of the tubes. By comparison, there are no visiblefluorescent bands in any of lanes 3, 5, and 7. This result is consistentwith the lack of amplification as indicated by visual analysis of thetubes.

[0223] With reference to FIG. 2B, all nucleic acid is fluorescentbecause it is stained with ethidium bromide. Therefore, the size markersin lanes 2 and 8 are now visible. Strong fluorescent bands having a sizeconsistent with the expected 111bp product are visible in lanes 4 and 6but are absent in lanes 3, 5 and 7. This data confirms production of theintended amplicon only in tubes 3 and 5 and further confirms that thenucleic acid was recovered from material originally electrostaticallybound to the Sepharose beads.

[0224] In summary, the data presented in FIGS. 1A and 1B, whenconsidered with the data presented in FIGS. 2A and 2B, conclusivelydemonstrates that it is possible to perform self-indicatingamplification assays wherein signal of a probe can be concentrated on amatrix to which an amplified target nucleic acid is electrostaticallyimmobilized.

[0225] Single Point Mutation Analysis

[0226] This experiment was used to examine whether or not it waspossible to achieve point mutation discrimination in the self-indicatingprobe-based assay. Unless otherwise stated, this experiment wasconducted essentially as described in part A, above, except that anunlabeled PNA oligomer (blocker probe) was added to achieve single pointmutation discrimination. Since control reactions not containing theblocker probe were performed, a comparison of the results obtained inthe presence and absence of blocker probe clearly demonstrates theremarkable improvement in target sequence identification resulting fromthe presence of the blocker probe. TABLE 5 Variable Factors Tube #PEI-Beads Template Blocker Probe 1 — — — 2 — pKRASWT — 3 — pKRASMU(31) —4 Sepharose — — 5 Sepharose pKRASWT — 6 Sepharose pKRASMU(31) — 7Sepharose — MU-15Blocker 8 Sepharose pKRASWT MU-15Blocker 9 SepharosepKRASMU(31) MU-15Blocker

[0227] PCR Materials & Methods:

[0228] This experiment comprised nine individual PCR reactions. Variablefactors examined within the set of nine reactions included the presenceor absence of PEI derivatized Sepharose beads (approximately 25,000PEI-Sepharose beads), the presence or absence of plasmid template(pKRASWT or pKRASMU(31) at 100 fmole per 50 μL reaction (0.4 nM)) andthe presence or absence of 400 pmole of PNA Blocker Probe (MU-15Blocker,See: Table 2). The total volume of the PCR reactions including glyceroland beads was 50 μL.

[0229] Table 5, below, summarizes the composition of various tubes withrespect to these variable factors. In addition, each PCR reactioncontained 45 pmole of the KRAS 5′ primer, 5 pmole of the KRAS3′ primer(See: Table 1), 3 mM MgCl2, 250 μM NTPs, 2.0 units AmpliTaq DNApolymerase, 50 mM KCl, 10 mM TRIS-HCl pH 8.3, 50 pmole BK.RAS-Cy3 (See:Table 2) non-nucleotide probe (Linear Beacon), 1.5 μL of glycerol and0.5 μL of Sepharose beads or water (control). The glycerol overlaid thebeads to temporarily shield the them from the reaction components in theearly stages of PCR so that the process would not be substantiallyinhibited by electrostatic binding of the primers to the matrix (beads)during the critical early thermocycles. During preparation and prior toPCR, the tubes were carefully handled to avoid mixing of components.

[0230] The PCR protocol involved a 20 second warm up to 95° C. (1stround only), followed by denaturing at 95° C. or 5 seconds, annealing at55° C. for 30 seconds, and extension at 74° C. for 30 seconds. Thedenaturation-annealing-extension cycle was repeated for 30 cycles.

[0231] Post PCR Workup/Analysis:

[0232] After PCR, tubes 1-9 were placed on a transilluminator tovisualize fluorescence and then photographed. FIG. 3 is a negative ofthe scanned image of the photograph taken of the nine mini-eppendorftubes immediately after PCR (tubes are labeled 1-9).

[0233] After photographing, the tubes were vortexed briefly, thencentrifuged briefly to concentrate the beads. The supernatants wereremoved and the beads were washed with 100 μL 50 mM NaCl, 100 mM TRIS-ClpH 7.6. The electrostatically bound nucleic acids were then releasedfrom the beads for analysis by vortexing in 10 μL of a solutioncontaining 100 mM CAPSO pH 10.7 and 2 M NaCl. The solution was thenseparated from the beads and 9 μL of each of the recovered solutions wascombined with 3 μL of 4× loading dye. A sample from each tube was thenrun on a 10-20% gradient gel to confirm nucleic acid amplification andidentify product size. FIGS. 4A and 4B are the negative of the images ofphotographs of the same 10-20% gradient polyacrylamide gel illuminatedon a UV transilluminator which were taken before (FIG. 4A) and after(FIG. 4B) ethidium bromide staining.

[0234] Results:

[0235] Tube Images/Photographs

[0236] With reference to FIG. 3, tube 1 was a negative controlcontaining no template and as expected, is not fluorescent after PCR(the tube contents resemble the background in the negative of theimage). However, the contents of tubes 2 and 3 were visibly fluorescentunder the transilluminator (darker than tube 1 in the negative of theimage). This was the expected result for tube 2, since amplification ofthe template should have produced the 111 bp amplicon containing atarget sequence to which the Linear Beacon is perfectly complementary.However, the amplicon generated in tube 3 contained a sequencecontaining a point mutation of the wild type amplicon. However, in theabsence of the blocker probe, the Linear Beacon probe will at leastpartially hybridize to the mutant amplicon generated from plasmidpKRASMU(31) under the hybridization conditions present since the mutantand wild type amplicons are so closely related. Partial hybridizationcauses enough fluorescent signal generation to be visible to the eyeunder the transilluminator. Since no Sepharose beads were present intubes 2 and 3, the orange color (darkness of the negative image) of thehybridized Linear Beacons is evenly distributed throughout the solution.Because the signal was not concentrated it did not produce a strongsignal in the Figure.

[0237] The reagent composition of tubes 4 through 6 are identical totubes 1 though 3, respectively, except that PEI-Sepharose beads werepresent during the PCR reaction. With reference to FIG. 3, tube 4 was anegative control containing no template and as expected, the solutionand Sepharose beads are not fluorescent after PCR as compared with tubes5 and 6 (the tube contents and beads resemble the background in thenegative of the image). With reference to tubes 5 and 6, the Sepharosebeads at the bottom of the tubes have become highly fluorescent with theintensity of tube 5 being slightly more intense as compared with tube 6.This result is as expected since amplification of the template shouldhave produced the 111 bp amplicon (electrostatically bound to the beadmatrix) to which the Linear Beacon hybridized to generate detectablesignal. The fluorescent intensity of tube 6 is lower since the LinearBeacon is not perfectly complementary to the amplicon but can at leastpartially hybridize to generate detectable signal under thehybridization conditions present. Because there is little differencebetween tubes 5 and 6, visual inspection of the tubes however, does notallow one to confirm whether or not the sample contained mutant or wildtype target sequence and is therefore not necessarily suitable forsingle point mutation analysis.

[0238] Upon comparison of tubes 2 and 3 with the intensity of signalfrom tubes 5 and 6, it becomes clear that concentration of thedetectable fluorescent signal on the beads allows one to more clearlydetect a positive result since the beads in tubes 5 and 6 are moreclearly positive as compared with the solutions in tubes 2 and 3.

[0239] The reagent composition of tubes 7 through 9 are identical totubes 4 though 6, respectively, except that in tubes 7-9, 400 pmole ofMU15Blocker probe was added prior to PCR amplification. With referenceto FIG. 3, tube 7 was a negative control containing no template and asexpected, the Sepharose beads are not fluorescent after PCR as comparedwith tubes 8 and 9 (the tube contents and beads resemble the backgroundin the negative of the image). Because the blocker probe is present,only the beads in tube 8 are clearly fluorescent as compared with thecontents of tubes 7 and 9. Thus, visual inspection of the tubes willallow one to confirm whether or not the sample contained mutant or wildtype target sequence. Therefore, this self-indicating assay is suitablefor single point mutation/discrimination analysis. It will beappreciated by those of ordinary skill in the art that quantitation ofdetectable signal can be achieved by using an instrument, such as a flowcytometer, and no more than routine experimentation.

[0240] In summary, the data presented in FIG. 3 indicates that it ispossible to perform homogeneous or closed tube amplification assayswherein signal of a probe can be concentrated on a matrix to which anamplified target nucleic acid is electrostatically immobilized.Furthermore, when utilizing blocker probes, the assay can be used forsingle point mutation analysis.

[0241] Gel Photographs/Images:

[0242] Analysis of the PCR reactions by gel was performed to determinethe presence and size of amplicons to thereby confirm that the visualanalysis of the tubes correlated with expected products of PCRamplification.

[0243] With reference to FIGS. 4A and 4B, the wells of the gel are atthe top of the photographs. The images in FIGS. 4A and 4B are notdirectly comparable since the photographs were made using differentexposure parameters. Aliquots of each tube were added near the top ofthe gel and electrophoretically directed towards the bottom of the gel.Lanes 1 and 12 contain two different double stranded DNA size markers;lane 1 is 100 bp ladder (New England BioLabs #323-1L) and lane 12 is a1000 bp ladder (New England BioLabs #323-2S). Band sizes are indicatedin FIG. 4B. Lanes 2-4 contain 9 μL of samples 1-3 respectively, lanes5-10 contain samples of released nucleic acid isolated from the beads intubes 4 through 9 respectively, and lane 11 is a blank.

[0244] With reference to FIG. 4A, the presence of inherently fluorescentbands can be seen in lanes 3, 4, 6, 7, and 9 (from samples 2, 3, 5, 6and 8 respectively) toward the bottom, and in the middle of the image.The fluorescent bands at the bottom of the image are most likely probemolecules which have migrated into the gel. The fluorescent bands in themiddle of the image can be attributed to the presence of the LinearBeacon still hybridized to the nucleic acid amplicon even after it hasmigrated into the gel. This result is consistent with amplification intubes 2, 3, 5, 6 and 8 as was indicated by the visual analysis andphotographing of the tubes. By comparison, there are no visiblefluorescent bands in any of lanes 2, 5, 8, and 10 (tubes 1, 4, 7 and 9).This result is consistent with the lack of fluorescence observed inthese tubes.

[0245] With reference to FIG. 4B, all nucleic acid is fluorescentbecause it is stained with ethidium bromide. Therefore, the size markersin lanes 1 and 12 are now visible. Strong fluorescent bands having asize consistent with the expected 111bp product are visible in lanes 3,4, 6, 7, 9 and 10 (tubes, 2, 3, 5, 6, 8 and 9) but are absent in lanes2, 5, and 8 (tubes 1, 4 and 7). This data confirms production of theintended amplicons in tubes 2, 3, 5, 6, 8 and 9 and further confirmsthat the nucleic acid was recovered from material originallyelectrostatically bound to the Sepharose beads. Most noteworthy is thepresence of a bands in both lanes 8 and 10 (tubes 7 and 9). These bandsconfirm that amplification occurred in these samples. Therefore the lackof signal in tube 9 as compared with tube 7 can only be attributable tothe presence of the blocker probe which allow one to achieve pointmutation discrimination of the amplicon.

[0246] In summary, the data presented in FIGS. 4A and 4B, whenconsidered with the data presented in FIG. 3, conclusively demonstratethat it is possible to perform self-indicating probe-based assayssuitable for single base discrimination (single point mutationdiscrimination) wherein signal of a probe can be concentrated on amatrix to which an amplified target nucleic acid is electrostaticallyimmobilized.

Example 13 Comparison of Assay Operating Range for PNA:DNA and DNA:DNAHybrids

[0247] This example is designed to compare the operating range forelectrostatic binding of non-nucleotide probes (e.g. PNA) with that ofthe most nearly equivalent nucleic acid probes in an electrostaticbinding assay for a nucleic acid target molecule which is nearlyequivalent in size to the probe. The goal is therefore to determine arange of ionic strength under which the non-nucleotide andpolynucleotide probes will bind to the matrix only if the nucleic acidtarget is present. For this example, a PNA probe (WT-15Flu PNA; SeeTable 2) and a DNA probe (WT-15Flu; See Table 1) was diluted in water toa concentration of 5 μM. The nucleic acid target (KRASWT(21); SeeTable 1) was also diluted in water to a concentration of 50 μM. TABLE 6Salt Buffers: Buffer ID Buffer Components: G 0 mM NaCl, 10 mM TRIS-Cl pH8.0 H 100 mM NaCl, 10 mM TRIS-Cl pH 8.0 I 200 mM NaCl, 10 mM TRIS-Cl pH8.0 J 300 mM NaCl, 10 mM TRIS-Cl pH 8.0 K 400 mM NaCl, 10 mM TRIS-Cl pH8.0 L 500 mM NaCl, 10 mM TRIS-Cl pH 8.0 M 600 mM NaCl, 10 mM TRIS-Cl pH8.0 N 700 mM NaCl, 10 mM TRIS-Cl pH 8.0

[0248] Next, four sets of eight eppendorf tubes were prepared with 100μL of each of the eight salt buffers described in Table 6. The four setsof tubes comprised the following experimental conditions: Into Set I wasadded the PNA probe but no nucleic acid target (KRASWT(21). This is the“no target” control. Into Set II was added the PNA probe and the nucleicacid target (KRASWT(21). Into Set III was added the DNA probe but nonucleic acid target (KRASWT(21). This is the “no target” control. IntoSet IV was added the DNA probe and the nucleic acid target (KRASWT(21).

[0249] These samples were prepared by adding one microliter of theappropriate stock of PNA probe or DNA probe to each tube in Sets I, II,III and IV to thereby achieve a final concentration of 250 nM probe. Toeach tube in Sets II and IV was also added one microliter of the stockof nucleic acid target (KRASWT(21) to thereby create a sample having afinal concentration of 2.5 μM target. To the “no target” control Sets Iand III, one microliter of water was added.

[0250] All tubes were vortexed briefly to mix.the ingredients, and heldat room temperature for approximately 5 minutes to allow hybridizationof the probes and targets. To each tube was then added one microliter ofPEI Sepharose particles suspended in water. The tubes were vortexedbriefly, allowed to stand at room temperature for approximately 5minutes, then centrifuged for 30 seconds to concentrate the particles atthe bottom.

[0251] Tubes were then arranged over a UV light source(transilluminator) and photographed. The negative image of thephotograph is presented as FIG. 5. Supernatants were then removed anddiscarded. Next, 100 μL of the appropriate salt buffer was added back tothe appropriate tubes. The PEI particles were resuspended in thehybridization buffer by vortexing vigorously and then each suspendedparticle sample was transferred to an individual well in a microtiterplate. The samples in the microtiter plate were immediately analyzed(Wallac, Victor, 1420 Multilabel Counter, Gaithersburg, Md.). Theresults of the analysis of fluorescence on the beads is presented inTable 7.

[0252] Results:

[0253] Tube Images/Photographs

[0254] With reference to FIG. 5, the four sets of tubes are arranged inorder from top to bottom. Within each set, the tubes are arranged inorder of increasing salt concentration from left to right. For example,the tube closest to the upper left comer of the Figure is Set I, BufferG (0.0 M NaCl), and the tube nearest the lower right comer is Set IV,Buffer N (0.7 M NaCl).

[0255] With reference to FIG. 5, Set I, the lack of fluorescent signalat the bottom of the tube indicates that the PNA probe has very littleaffinity for the particles in the absence of the nucleic acid target(KRASWT(21). In Set II by comparison, the PNA probe is concentrated onthe matrix to a salt concentration of approximately 300 mM (See SaltBuffer J). This result is consistent with hybridization of the probe tothe target sequence electrostatically bound to the matrix. The lack ofprobe concentrated on the matrix at salt concentrations above 300 mM islikely due to the lack of binding of the short nucleic acid target(KRASWT(21) at those salt concentrations. Taken as a whole, the dataindicates that the PNA probe does not interact with the matrix under anyconditions of ionic strength examined. Thus, the applicable range forthe assay utilizing this PNA probe is at least 0-700 mM salt.

[0256] With reference to FIG. 5, Set III, the DNA probe is substantiallyconcentrated on the matrix up to a salt concentration of approximately300 mM (See Salt Buffer J) and weakly up to a salt concentration of 400mM (See Salt Buffer K). This data indicates that the native DNA probehas a substantial inherent affinity for the matrix. By comparison, SetIV, indicates that the probe/target sequence hybrid raises the presenceof probe strongly concentrated on the matrix up to a salt concentrationof approximately 400 mM (See Salt Buffer K) and weakly up to a saltconcentration of 500 mM (See Salt Buffer L). Therefore the operatingrange for discriminating probe from probe/target sequence complex whenusing this all DNA system is approximately 300 to 500 mM salt. This avery narrow operating range by comparison with the PNA probe. Note: Theapparent conflict between the results of Experiment 9 and the resultsdescribed above, wherein the PNA probe WT-15Flu detectably binds to thematrix up to 100 mM salt (Exp. 9) but does not interact with the supporteven in 0 mM salt, has been confirmed to be condition dependent. Thebuffer in Experiment 9 contains Tween-20 which appears to promote theinteraction of the PNA probe with the support. Additionally, the PNAprobe in this experiment was first added to water from the concentratedstock of 1/1 DMF:water which appears to decrease the interaction of thePNA probe with the support. To avoid doubt, all data is consistent withthe fluorescein label being the primary source of interaction with thematrix which was an apparent result of Experiment 9.

[0257] Quantitation of Particle Associated Fluorescence

[0258] The visual comparison of the tube was also confirmed byquantitative analysis of fluorescence of the resuspended beads. Thequantitative fluorescence measurements as well as derived data for thebeads is presented in Table 7.

[0259] With reference to Table 7, the raw fluorescent reading from eachsample (Sets I-IV; rows B-E, respectively) of suspended particles ateach of the salt buffers (Buffers G-N; columns 2-9, respectively) ispresented. From this data the signal to noise data for PNA probe (row F)and DNA probe (row G) is mathematically derived from the rawfluorescence data. For example, the raw fluorescent value obtained forBuffer G in Set I (column 2, row B=738 rlu) was divided into the rawfluorescent value of Buffer G in Set II (column 2, row C=12736 rlu) toobtain the S/N value for the PNA probe in Buffer 0 of 17.3 rlu(12736÷738=17.3 (column 2 row F)). TABLE 7 Bead Fluorescence Data 1 2 34 5 6 7 8 9 A Buffer# G H I J K L M N B Set I 738 652 618 956 696 1052992 1124 C Set II 12736 12052 11212 8610 1726 830 420 660 D Set III36422 39807 33577 28001 7572 5750 5286 4640 E Set IV 36170 47133 4657043731 43495 13020 2284 1710 F PNA S/N 17.3 18.5 18.1 9.0 2.5 0.8 0.4 0.6G DNA S/N 1.0 1.2 1.4 1.6 5.7 2.3 0.4 0.4

[0260] The signal to noise ratio calculated from the quantitative rawfluorescence data for the PNA probe agrees with the visual analysis. Astrong signal can be detected above the background from 0-300 mM salt(See: row F, columns 2-5). By comparison, only a weak signal is detectedfor the DNA probe at salt concentrations of between 300 and 500 mM (See:row G. columns 5-7).

[0261] Taken as a whole the visual data of FIG. 5 and the quantitativedata of Table 7 clearly demonstrates that the non-nucleotide PNA probesoperate within a substantially greater range of salt concentrations ascompared with the most nearly equivalent DNA probes when used in anelectrostatic immobilization assay. The PNA probes also provide asubstantially greater signal to noise ratio as compared with the DNAprobes. Consequently, the data indicates several advantages which makethe PNA probes the superior choice for performing probe-based analysisof nucleic acid electrostatically immobilized to a matrix.

Example 14 Single Point Mutation Discrimination Using aProtection/Digestion Assay

[0262] This experiment was designed as a Protection/Digestion Assaysuitable for single point mutation discrimination. As designed, theassay also demonstrates a means for improving the assay caused byadjusting the temperature of the assay to a point where non-specifichybrids begin to melt and the nucleic acid which thereby causes thenon-specific signal now becomes available as a substrate to the enzyme.Digestion of the interfering non-target sequence results in asubstantial improvement in signal to noise ratio of the assay. The assaywas also substantially simplified by use of a self-indicating LinearBeacon (BK.RAS-Cy3; See Table 2) and electrostatic immobilization of theLinear Beacon/target sequence hybrid to Sepharose particles whichenabled the rapid electrostatic capture and quantitation of the LinearBeacon/target sequence hybrid.

[0263] Materials:

[0264] Mung Bean Nuclease and 10× buffer were obtained from New EnglandBioLabs. The enzyme is supplied at 10 units per microliter. When the 10×buffer is diluted according to the manufactures instructions, the buffercontains 50 mM sodium acetate, 30 mM sodium chloride, 1 mM zinc chlorideand has a pH of 5.0 at 25° C.

[0265] Experimental:

[0266] This experiment comprised 6 samples in which the PNA probe washybridized to either the KRASWT(24) target (See: Table 1) or the singlebase mismatch, KRASMU(24) target (See: Table 1). A no target control andcontrol samples without enzyme were also performed. The assay wasperformed at 65° C. This temperature is below the Tm of the perfectcomplement (BK.RAS-Cy3/KRASWT(24)) which has been measured to beapproximately 81° C. and very close to the Tm of the imperfectcomplement (BK.RAS-Cy3/KRASMU(24)) which has been measured to beapproximately 67° C. under identical conditions. This temperature iswithin the range of five degrees above and ten degree below the meltingtemperature of the imperfect complement which is being discriminated inthe assay and which has a single point mutation as compared with thetarget sequence KRASWT(24). The composition of the six samples issummarized below:

[0267] Sample 1. KRASWT(24) target,+enzyme

[0268] Sample 2. KRASMU(24) target,+enzyme,

[0269] Sample 3. No Target+enzyme

[0270] Sample 4. KRASWT(24) target, no enzyme

[0271] Sample 5. KRASMU(24) target, no enzyme,

[0272] Sample 6. No Target, no enzyme

[0273] For this experiment, PNA probes and DNA targets were added to afinal concentration of 0.33 μM in a 100 μL volume of 1× mung beannuclease buffer. Samples were heated to 95° C. for 5 minutes to denaturehybrids and then cooled to 65° C. After 5 minutes of equilibration at65° C., samples 1-3 were treated with 0.3 μL mung bean nuclease. Samples4, 5 and 6 were not treated with nuclease. All samples were vortexedbriefly, then allowed to incubate for 10 minutes at 65° C. After theincubation, all samples were treated with 1 μL of PEI Sepharoseparticles, vortexed vigorously, then centrifuged for 30 seconds topellet the particles. Supernatants were removed and the particles wereresuspended in 100 μL 1× mung bean nuclease buffer. The entire contentsof each tube was transferred to a microtiter plate and analyzed forfluorescence using a Wallac, Victor, 1420 Multilabel Counter.Fluorescence values are described in relative light units (rlu).

[0274] Results:

[0275] Fluorescent measurements of the two “no target” controls, sample#3 and sample #6, gave similar values, as would be expected (400 and 466rlu respectively). The “no target” values were subtracted from the rawfluorescent values of the other samples to obtain values minusbackground signal. The values minus background signal for the remainingsamples were as follows; sample #1, (4926 rlu); sample #2, (338 rlu);sample #4, (8896 rlu); and sample#5, (2532 rlu).

[0276] Comparison of enzyme treated and untreated samples reveals therelative benefits of nuclease treatment. Comparison of sample #1 andsample #4 demonstrates a 45% loss of signal from the complimentarytarget, KRASWT(24), when treated with the nuclease((8896-4929)÷8896=45%). In contrast, comparison of sample #2 with sample#5 demonstrates an 87% loss of signal from the single base mismatchtarget, KRASMU(24), from enzyme treatment ((2532-338)÷2532)=87%). As aresult of the differential loss in signal from the imperfect complementas compared with the perfect complement, which is attributable toenzymatic digestion, there is a corresponding increase in signal tonoise ratio (fully complimentary signal divided by mismatch signal, S/N)for the assay. The S/N value for the sample which was not treated withenzyme was 3.5 (8896÷2532=3.5) and the S/N value for the sample whichwas treated with enzyme was 14.6 (4926÷338=14.6). Though a loss ofspecific signal was observed in the enzyme treated samples (compare rawfluorescence for sample #'s 1 and 2 with 4 and 5, respectively), the netgain in signal to noise was very beneficial to the overall performanceof the assay.

[0277] Taken as a whole, this data demonstrates that theProtection/Digestion Assay can be combined with electrostaticimmobilization of the non-nucleotide probe/target sequence complex toprovide a rapid result. The non-nucleotide probe can be a Linear Beaconand the assay self-indicating. Additionally, the result of the assay canbe substantially enhanced by judicious modulation of assay temperatureto thereby melt and digest nucleic acid which caused false positiveresults.

Example 15 Array Assay

[0278] For this assay, a commercially available microscope slide havinga cationic surface was used to electrostatically immobilize premixedsamples containing nucleic acid and probe which had been deposited onthe slide into an array of spots. The microscope slide was then washedto remove unhybridized probe and detect the target sequence if presenton the microscope slide.

[0279] Preparation of Probe, Targets, and Particles:

[0280] The cyanine-3 (Cy3) labeled PNA 15-mer (RASWT-Cy3), in a solutionof 50% aqueous N,N-dimethylformamide at a concentration of 570 pmol/μL,was diluted to a concentration of 20 pmol/μL in hybridization buffer(12% aqueous formamide, 5 mM Tris hydrochloride, 25 mM sodium chlorideand 0.05% SDS at a pH of 7.5). The DNA oligonucleotide 31-mer(KRASMU(31)), that was complementary to RASWT-Cy3, in water at aconcentration of 20 pmol/μL was diluted 221 fold to a concentration of 1pmol/μL in hybridization buffer. The DNA oligonucleotide 60-mer(CompDNA), that was non-complementary to RASWT-Cy3, in water atconcentration of 90 pmol/μL, was diluted 90 fold to a concentration of 1pmol/μL in hybridization buffer.

[0281] Hybridization, Spotting and Data Acquisition:

[0282] Tube A: In a microfuge tube was combined 1 μL of water with 1 μLof RASWT-Cy3 and 18 μL of hybridization buffer.

[0283] Tube B: In a microfuge tube was combined 1 μL of KRASMU(31) with1 μL of RASWT-Cy3 and 18 μL of hybridization buffer.

[0284] Tube C: In a microfuge tube was combined 1 μL of CompDNA with 1μL of RASWT-Cy3 and 18 μL of hybridization buffer.

[0285] Tubes A, B and C were incubated for 15 min at room temperatureand then 0.2 μL of the solution from each tube was applied as a row ofdroplets to a GAPS Coated Slide (Corning, Corning N.Y.). The slide had agamma-aminopropyl silane coated surface. The slide was placed, for aperiod of approximately 20 min, in an oven maintained at 50° C. untilthe spots had dried. The slide was cooled to room temperature and imagedto verify the location of the spots on the slide. A Genetic Microsystemsarray microimager,(GMS 318, Woburn, Mass.) was used to acquire slideimages using the green laser according to the manufacturer'sinstructions. The slide was then removed from the imager and washed withhybridization buffer in a small tray with gentle agitation for 5 min atroom temperature. The slide was then rinsed with deionized water, shakento remove excess water, and allowed to dry on the bench. The image ofthe washed slide was again acquired.

[0286] Results:

[0287]FIGS. 6A and 6B are the images of the slide taken before and afterthe wash step, respectively. Prior to washing there were three visiblespots labeled A, B & C, corresponding to the reactions from Tubes A, B &C. However, after the wash step (FIG. 6B), Spot A was no longer visibleto the instrument. This demonstrates that the PNA probe, in the absenceof any nucleic acid, was removed from the slide surface by the washstep. When the complementary target KKRASMU(31) was present, the visiblesignal at the array location was retained (Spot B), presumably due tohybridization of the probe to the electrostatically immobilized target.When a noncomplementary nucleic acid was used, most of the PNA probe waswashed away (Spot C). Taken as a whole, the data demonstrates that it ispossible to prepare a matrix array of electrostatically immobilizednucleic acid and easily assay for the presence of a target sequencelocated thereon using a non-nucleotide probe.

Example 16 Line Assay

[0288] In the example, a line assay is performed wherein anon-nucleotide probe/target sequence complex is captured using a line ofpolycationic polymer on a commercially available membrane materialwherein reagents are allowed to wick into the membrane as is typical ofa lateral flow assay.

[0289] Preparation of Membrane:

[0290] Strips (2.5 cm×20 cm) of Millipore membrane (P/N WOPP, Bedford,Mass.) were wet in a solution of 0.5% glutaraldehyde in ethanol. The wetstrips were placed on a piece of Whatman 3MM paper in a fume hood. After3 minutes, when the filter strips appeared dry, they were removed fromthe hood and place on the platen of an Ivek microstriper (Ivek Corp.,Springfield, Vt.). A 3 mm wide line of polyethylenamine (PEI) solutionwas then applied along the midpoint of each membrane strip. The PEIsolution was previously prepared by dissolving 750,000 molecular weightPEI (Aldrich Chemical, Milwaukee, Wis.) in water and adjusting the pH to8.5 with dilute hydrochloric acid. The PEI solution was then diluted toa final concentration of 1 mg of PEI per milliliter.

[0291] Once the filter strips were striped with the PEI solution, theywere allowed to dry overnight on the bench. The next day the strips werewashed with dilute hydrochloric acid, pH ˜3, for 45 minutes. The stripswere then washed with water and placed on Whatman 3MM paper to dry for24 hrs. The strips were cut into smaller pieces 1 cm wide by 2.5 cm longsuch that each strip had a PEI line across its mid point. At one end, 5mm of each piece was sandwiched between two pieces of Whatman 3MM paper(2×1 cm) using a small metal Bulldog clamp.

Preparation of Probe, Targets, and Particles

[0292] A biotinylated PNA 15-mer (BioP-15), in a solution of 50% aqueousN,N-dimethylformamide at a concentration of 333 pmol/μL, was diluted 333fold to final concentration of 1 pmol/μL into hybridization buffer (50%aqueous formamide, 20 mM Tris hydrochloride, 100 mM sodium chloride and0.1% SDS, pH of 7.5). The DNA oligonucleotide 60-mer (CompDNA; See Table1), complementary to BioP-15 in water at a concentration of 90 pmol/μL,was diluted 90 fold to a concentration of 1 pmol/μL in hybridizationbuffer. A DNA oligonucleotide 60-mer (NonCompDNA; See Table 1) that wasnon-complementary to BioP-15, in water at a concentration of 221pmol/μL, was diluted 221 fold to a concentration of 1 pmol/μL inhybridization buffer. A suspension of streptavidin gold particles(Arista Biologicals, Inc., Bethlehem, Pa.)), 40 nm diameter, was diluted20-fold with hybridization buffer.

[0293] Hybridization:

[0294] Tube A: In a microfuge tube was combined 2.5 μL of CompDNA with2.5 μL of BioP-15. The reaction was then incubated for 2 min at roomtemperature and 20 μL of 40 nm streptavidin gold particles inhybridization solution was added.

[0295] Tube B: In a microfuge tube was combined 2.5 of μL NonCompDNAwith 2.5 μL of BioP-15. The reaction was then incubated for 2 min atroom temperature and 20 μL of 40 nm streptavidin gold particles inhybridization solution was added.

[0296] Line Assay:

[0297] Tubes A and B were then incubated for 15 min. at room temperatureafter addition of the gold particles. The contents of the tubes weretransferred onto a small piece of Parafilm lab film (American CanCompany). Onto different pieces of Parafilm were spotted two 20 μldroplets of hybridization buffer. Into each drop was dipped the end of amembrane strip such that the buffer wicked towards the end held by theBulldog clamp. The filter strips were held in contact with the liquiduntil entire droplet had wicked into the membrane. The ends of the twostrips were then dipped separately into the contents of Tube A or Tube Bthat had previously been transferred to clean sections of the lab film.Once the entire A and B droplets had been wicked into their respectivefilter strips, the filter ends were then separately dipped into 10 μLdroplets of hybridization buffer.

[0298] Results:

[0299] In the case of Tube A that contained the BioP-15 and its DNAcomplement CompDNA, a red line formed across the filter strip during thewicking of the Tube A contents into the filter strip. In the case ofTube B, no line was seen. The results demonstrate the feasibility of asimple line assay for detecting nucleic acids using a cationic polymeras a capture zone on the membrane filter.

[0300] Equivalents

[0301] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention as defined by the appended claims. Those skilled in theart will be able to ascertain, using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed in the scope of the claims.

1 15 1 24 DNA Artificial Sequence misc_feature (1) 5′ biotin label 1gtggtagttg gagctggtgg cgta 24 2 24 DNA Artificial Sequence misc_feature(1) 5′ biotin label 2 gtggtagttg gagcttgtgg cgta 24 3 31 DNA ArtificialSequence misc_feature (1) 5′ biotin 3 gtggtagttg gagcttgtgg cgtaggcaag a31 4 15 DNA Artificial Sequence misc_feature (1) 5′ fluorescein label 4acgccaccag ctcca 15 5 15 DNA Artificial Sequence misc_feature (1) 5′fluorescein label 5 acgccacaag ctcca 15 6 20 DNA Artificial SequenceDescription of Artificial Sequencesynthetic probe, primer or target 6gcttgtttcg gcgtgggtat 20 7 20 DNA Artificial Sequence Description ofArtificial Sequencesynthetic probe, primer or target 7 taggttgaggccgttgagca 20 8 20 DNA Artificial Sequence Description of ArtificialSequencesynthetic probe, primer or target 8 atgactgaat ataaacttgt 20 920 DNA Artificial Sequence Description of Artificial Sequencesyntheticprobe, primer or target 9 ctctattgtt ggatcatatt 20 10 60 DNA ArtificialSequence Description of Artificial Sequencesynthetic probe, primer ortarget 10 tcactagtcc cttcaaggct agcagtataa tgggttctag gtaaacgttccaccgttact 60 11 60 DNA Artificial Sequence Description of ArtificialSequencesynthetic probe, primer or target 11 agtaacggtg gaacgtttacctagaaccca ttatactgct agccttgaag ggactagtga 60 12 111 DNA ArtificialSequence Description of Artificial Sequencesynthetic probe, primer ortarget 12 atgactgaat ataaacttgt ggtagttgga gcttgtggcg taggcaagagtgccttgacg 60 attcagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga g111 13 111 DNA Artificial Sequence Description of ArtificialSequencesynthetic probe, primer or target 13 atgactgaat ataaacttgtggtagttgga gctggtggcg taggcaagag tgccttgacg 60 attcagctaa ttcagaatcattttgtggac gaatatgatc caacaataga g 111 14 111 DNA Artificial SequenceDescription of Artificial Sequencesynthetic probe, primer or target 14ctctattgtt ggatcatatt cgtccacaaa atgattctga attagctgta tcgtcaaggc 60actcttgcct acgccaccag ctccaactac cacaagttta tattcagtca t 111 15 111 DNAArtificial Sequence Description of Artificial Sequencesynthetic probe,primer or target 15 ctctattgtt ggatcatatt cgtccacaaa atgattctgaattagctgta tcgtcaaggc 60 actcttgcct acgccacaag ctccaactac cacaagtttatattcagtca t 111

We claim:
 1. A kit for the analysis of a sample containing a nucleicacid molecule comprising a target sequence, said kit comprising a matrixand at least one non-nucleotide probe having a probing nucleobasesequence that sequence specifically hybridizes, under suitablehybridization conditions, to at least a portion of the target sequencesought to be detected in said sample to thereby form a non-nucleotideprobe/target sequence complex and wherein the backbone of thenon-nucleotide probe or probes is sufficiently neutral or positivelycharged, under electrostatic binding conditions, that it exhibits littleor no affinity for the matrix.
 2. The kit of claim 1, further comprisingone or more reagents suitable for modulating the electrostatic bindingconditions of the assay.
 3. The kit of claim 1, further comprisingenzymes that degrade sample contaminants but not the non-nucleotideprobe/target sequence complex.
 4. The kit of claim 1, wherein thecomponents of the kit are selected to detect organisms in food,beverages, water, pharmaceutical products, personal care products, dairyproducts or environmental samples.
 5. The kit of claim 1, wherein thecomponents of the kit are selected to test raw materials, products orprocesses.
 6. The kit of claim 1, wherein the components of the kit areselected to examine clinical samples such as clinical specimens orequipment, fixtures and products used to treat humans or animals.
 7. Thekit of claim 1, wherein the components of the kit are selected to detecta target sequence that is specific for a genetically-based disease or isspecific for a predisposition to a genetically-based disease.
 8. The kitof claim 1, wherein the components of the kit are selected detect atarget sequence in a forensic technique such as prenatal screening,paternity testing, identity confirmation or crime investigation.
 9. Thekit of claim 1, wherein the components of the kit are selected toperform a homogeneous assay.
 10. The kit of claim 1, wherein the matrixis provided in a form suitable for performing a lateral flow assay. 11.The kit of claim 1, wherein the matrix is provided in a form suitablefor performing a line assay.
 12. The kit of claim 1, wherein the kitcomprises at least two independently detectable non-nucleotide probesand comprises components selected to perform a multiplex assay.
 13. Akit for the analysis of a sample containing a nucleic acid moleculecomprising a target sequence, said kit comprising a matrix and at leastone non-nucleotide “Beacon” probe having a probing nucleobase sequencethat sequence specifically hybridizes, under suitable hybridizationconditions, to at least a portion of the target sequence sought to bedetected in said sample to thereby form a non-nucleotide “Beacon”probe/target sequence complex and wherein the backbone of thenon-nucleotide probe or probes is sufficiently neutral or positivelycharged, under electrostatic binding conditions, that it exhibits littleor no affinity for the matrix.
 14. The kit of claim 13, furthercomprising one or more reagents suitable for modulating theelectrostatic binding conditions of the assay.
 15. The kit of claim 13,further comprising enzymes that degrade sample contaminants but not anon-nucleotide probe/target sequence complex.
 16. The kit of claim 13,wherein the components of the kit are selected to detect organisms infood, beverages, water, pharmaceutical products, personal care products,dairy products or environmental samples.
 17. The kit of claim 13,wherein the components of the kit are selected to test raw materials,products or processes.
 18. The kit of claim 13, wherein the componentsof the kit are selected to examine clinical samples such as clinicalspecimens or equipment, fixtures and products used to treat humans oranimals.
 19. The kit of claim 13, wherein the components of the kit areselected to detect a target sequence that is specific for agenetically-based disease or is specific for a predisposition to agenetically-based disease.
 20. The kit of claim 13, wherein thecomponents of the kit are selected to detect a target sequence in aforensic technique such as prenatal screening, paternity testing,identity confirmation or crime investigation.
 21. The kit of claim 13,wherein the components of the kit are selected to perform a homogeneousassay.
 22. The kit of claim 13, wherein the kit comprises at least twoindependently detectable non-nucleotide “Beacon” probes and comprisescomponents selected to perform a multiplex assay.
 23. The kit of claim22, wherein the components are selected to perform a multiplexself-indicating assay.
 24. The kit of claim 13, wherein the matrix isprovided in a form suitable for performing a lateral flow assay.
 25. Thekit of claim 13, wherein the matrix is provided in a form suitable forperforming a line assay.